Richard A Cloney 1984

University of Seattle

and Friday Harbor Laboratories

Manual on fixation and osmification procedures for TEM analysis of marine invertebrate larvae

Document handed out at courses run at the FHL in the 1980ties and amended by JT Hoeg handwritten notes

For free distribution with source indicated

Jens T Hoeg

Marine Biology Section Department of Biology

University of Copenhagen Universitetsparken 5

DK-2100 Copenhagen Denmark jthoegbiokudk

J J

PHOSPHATE BUFFEHED GLUTARALDEHYDE recommended for a variety of marIne organIsms (HL Dunlap 1965 RA Cloney and E Florey 1968)

A Basic formula of primary fixative

Final concentrations Nilliosmoles

25 Glutaraldehyde 275 02M Nillonigs phosphage buffer (pH 76) 420 014N Sodium chloride 275

970

II Stock SolutlOns

a) Glutaraldehyde solutions are available in concentrations of 70 50 25 and 8 These solutions should be stored in a refrigerator at about 50 C until ready for use

For dilution use the following for~u1a

Voll x Concl = Vo12 x Conc2

Example How much water IS needed to make 25 from 70 glutaraldehyde

2ml x 70 Vo12 x 25

(Divide both sides of equation by 25)

56ml = Vo12

For a 25 solution dilute 2ml of a 70 solution to 56ml with water

b) Millonigs phosphate buffer (04M)

Sodium phosphate (monobasic) 1108 ms Sodium hydroxide 285 gms Distilled water to make 20000 ml

The pH should be about 76 after amp dilution with water to 02M The osmolality should be about 420 aftp[ dilution

c) Salt solution (034M)

~odium chlorid MW 5845 034M x 5845 r 9--87 -gmliter

397 gm200 ml

Add water to make 1 liter or 200 ml

It is convenient to have available a 1OM solution of sodium chloride (5845 gmliter) if you wish to modify the o~molality of the basic fixitive It can be diluted to the appropriate strength and used in place of the 034M solution recommended in the basic formula ~en an 8 solution of glutaraldehyde is used it is necessary to use a stock solution of 0747M sodium chloride in order to end up with the appropriate final concentrations of the three components

c Preparation of fixative from stock solutions using 25 glutaraldehyde

250 Glutaraldehyde 5 ml 034M Sodium chloride 20 ml 04M Phosphate bu f fe r 25 ml

50 ml

Preparation of fixative from stock solutions using 8 glutaraldehyde

80 Glutaraldehyde 10 ml 075M Sodium chloride 6 ml O4r Phospbate bu f fe r 16 ml

32 ml

The final solution of fixative should have an osmolality of 970 if all of the solutions have been properly prepared and mixed in the correct proportions It is advisable to check the osmolality with an osmometer The osmolality of seawater at the Friday Harbor Laboratories is usually about~but varies with the season ~

Fdmary poundxoo prp Vi 1J ( 0 d d 0D

The glutaraldehyde must penetrate the tissue before it can react with and stabilize the organic compounds in the cells and intercellular substances Small organisms (lmm in thickness) can be placed directly in the fixative Sometimes larger organisms or pieces of tissue can be adequately fixed without reducing their size but it is a good practice to fix pieces of several different sizes (See note under secondary fixation)

2

Fix the tissue for about 1 hour at room temperature Longer and shorter times can be tried Some specimens seem to be undamaged after as long as several days in the fixative Some small specimens (eggs protozoans) can be fixed in 15 minutes or less After primary fixation in glutaraldehyde the tissue may be rinsed in a phosphate buffer solution for 10-15 minutes or alternatively the tissue can be transferred directly into a small amount of the secondary fixative (see below) for a few seconds to dilute out the glutaraldehyde solution

Phosphate Buffer Rinsing Solution (optional)

Mix equal parts of 04M phosphate buffer stock solution and 06M sodium chloride

Final concentrations

02M Phosphate buffer pH 76 420 moms 03M Sodium chloride 560 moms

E Secondary fixation

Secondary fixation in Os04 is necessary to stabilize the lipids and improve the contrast for electron microicopy Either phosphate or bicarbonate buffered Os04 can be used I (RAC) prefer bicarbonate as a buffer for ascidians and mollusks

Phosphate buffered Os04 Final concentrations

40 Osmium tetroxide U5 ml Osmium tetroxid( 04M Phosphate buffer 05 ml 02M Phosphate

(pH 76)

Bicarbonate buffered Os04 Final concentrations

40 Osmium tetroxide 05 ml 20 25 Sodium bicarbonate 05 ml 125

(0625 gm2S ml water)

Adjust the pH of the bicarbonate buffer to 72 with HCl just before mixing with the Os04 The pH will rise to about 74 when it IS

diluted with Os04

The period of secondary fixation should be between ~ and 1 hour at room temperature depending on the size of the pieces of tissue Os04 penetrates much more slowly than glutaraldehyde If large (thicker than 1 mm) pieces have been used for primary fixation it would be advisable to cut them up before placing them in the secondary fixative Dehydrate and embed the tissues as usual

3

STEVE TORRENCES FIXATIVE

For marine embryos and larval tissues (especially ascidians)

A Primary fixative

approx osmolality

_______ 2 0 Glutaraldehyde 200

zfgtC 02M -shySodium cacodylate 375

0275M Sucrose 300

1 0 Tannic acid 85

960

B Preparation of fixative

Combine the following according to recommendation in section D

r80 Glutaraldehyde 100 ml J-ff 2 ~

Isj()O8M Sodium cacodylate 100 ml ~+--

055M Sucrose 200 ml

1annic acid powder 040 gm

cent(56- - 0--- tit Y17 r--shyC Stock solution information

c S -teth-Sodium cacodylate MW = 214

08 x 214 = 1712 1712 gm + 100 ml water 08M solution

[ Sucrose MW 3423 )

055 x 3423 = 1882 1882 gm + 100 ml water 0S5M solution

D Combining reagents

Tannic acid breaks down slowly in the light The powder needs to be protected from exposure to light It should be added to the fixative just before use Adjust the pH to 74 with 02N HCl after all components (including the tannic acid) have been combined The final osmolality should be about 960

4

E

F

( ~

G

H

Primary fixation methodology

Pre-cool the fixative and fix for 2-3 hours on ice Rinse the specimens briefly with a small amount of the fixative at the beginning or fix in a large volume of fixative since otherwise the calcium in the sea water may cause the tannic acid to precipitate

Buffer rinse

Rinse the tissues 3 or 4 times in the following solution to ensure removal of excess tannic acid Tannic acid precipitates with osmium tetroxide This solution should also be cooled on lce

1- 08M Sodium cacodylate 1 part 21M0fCtS-t(0 ~I

055M Sucrose 2 part (037H) I=- ~~x

The final concentrations are listed ln parentheses 1 M 1- ( Jl C

I~

p f J M j r C rD$( 01 ltgt

OSlt~ ( 61 ~ Second~y- ~ative J (N

40 Osmium tetroxide 1 part (2) 25 Sodium bicarbonate 1 part 025) ~

Adjust the pH of the bicarbonate buffer to 72 with Hel just before mixing it with Os04 Pre-cool the fixative on lce Fix for 1-2 hours on lce It may be necessary to rinse the specimens once with this fixative at the beginning to remove the last of the tannic acid if a brown precipitate forms I rr rl(t IMc Jo tL

IT vG-4te-J ( f~e-If c JDehydration infiltration and embedding

L -rtJ 6 J La-fS (1Ltdc1t r~cy ) Dehydration should be carried out on ice through the 90 step nit j

specimens should be brought to room temperature in the first chan ~ e of absolute solvent The best results are obtained when acetone or isopropanol are used as dehydrating agents and as antemedia

If ethanol is used as a dehydrating agent it must be followed by propylene oxide as an antemedium

Dr Steven Torrence took Zoology 583 several years ago He investigated the fine structure of some post-larval ascidians that attach to solid surfaces such as rocks wood and plastic Petri dishes He initially fixed detached specimens in bicarbonate buffered osmium tetroxide in combination with the standard Epon method The results were acc e ptable but not outstanding Later he modified his technique by omitting propylene oxide so that he could fix animals that were attached to plastic Petri dishes (Propylene oxide dissolves these dishes) Even though he used the same exact techniques repeatedly his results were extremely poor He could not obtain results that were even close to those obtained in his initial studies

5

J J

PHOSPHATE BUFFEHED GLUTARALDEHYDE recommended for a variety of marIne organIsms (HL Dunlap 1965 RA Cloney and E Florey 1968)

A Basic formula of primary fixative

Final concentrations Nilliosmoles

25 Glutaraldehyde 275 02M Nillonigs phosphage buffer (pH 76) 420 014N Sodium chloride 275

970

II Stock SolutlOns

a) Glutaraldehyde solutions are available in concentrations of 70 50 25 and 8 These solutions should be stored in a refrigerator at about 50 C until ready for use

For dilution use the following for~u1a

Voll x Concl = Vo12 x Conc2

Example How much water IS needed to make 25 from 70 glutaraldehyde

2ml x 70 Vo12 x 25

(Divide both sides of equation by 25)

56ml = Vo12

For a 25 solution dilute 2ml of a 70 solution to 56ml with water

b) Millonigs phosphate buffer (04M)

Sodium phosphate (monobasic) 1108 ms Sodium hydroxide 285 gms Distilled water to make 20000 ml

The pH should be about 76 after amp dilution with water to 02M The osmolality should be about 420 aftp[ dilution

c) Salt solution (034M)

~odium chlorid MW 5845 034M x 5845 r 9--87 -gmliter

397 gm200 ml

Add water to make 1 liter or 200 ml

It is convenient to have available a 1OM solution of sodium chloride (5845 gmliter) if you wish to modify the o~molality of the basic fixitive It can be diluted to the appropriate strength and used in place of the 034M solution recommended in the basic formula ~en an 8 solution of glutaraldehyde is used it is necessary to use a stock solution of 0747M sodium chloride in order to end up with the appropriate final concentrations of the three components

c Preparation of fixative from stock solutions using 25 glutaraldehyde

250 Glutaraldehyde 5 ml 034M Sodium chloride 20 ml 04M Phosphate bu f fe r 25 ml

50 ml

Preparation of fixative from stock solutions using 8 glutaraldehyde

80 Glutaraldehyde 10 ml 075M Sodium chloride 6 ml O4r Phospbate bu f fe r 16 ml

32 ml

The final solution of fixative should have an osmolality of 970 if all of the solutions have been properly prepared and mixed in the correct proportions It is advisable to check the osmolality with an osmometer The osmolality of seawater at the Friday Harbor Laboratories is usually about~but varies with the season ~

Fdmary poundxoo prp Vi 1J ( 0 d d 0D

The glutaraldehyde must penetrate the tissue before it can react with and stabilize the organic compounds in the cells and intercellular substances Small organisms (lmm in thickness) can be placed directly in the fixative Sometimes larger organisms or pieces of tissue can be adequately fixed without reducing their size but it is a good practice to fix pieces of several different sizes (See note under secondary fixation)

2

Fix the tissue for about 1 hour at room temperature Longer and shorter times can be tried Some specimens seem to be undamaged after as long as several days in the fixative Some small specimens (eggs protozoans) can be fixed in 15 minutes or less After primary fixation in glutaraldehyde the tissue may be rinsed in a phosphate buffer solution for 10-15 minutes or alternatively the tissue can be transferred directly into a small amount of the secondary fixative (see below) for a few seconds to dilute out the glutaraldehyde solution

Phosphate Buffer Rinsing Solution (optional)

Mix equal parts of 04M phosphate buffer stock solution and 06M sodium chloride

Final concentrations

02M Phosphate buffer pH 76 420 moms 03M Sodium chloride 560 moms

E Secondary fixation

Secondary fixation in Os04 is necessary to stabilize the lipids and improve the contrast for electron microicopy Either phosphate or bicarbonate buffered Os04 can be used I (RAC) prefer bicarbonate as a buffer for ascidians and mollusks

Phosphate buffered Os04 Final concentrations

40 Osmium tetroxide U5 ml Osmium tetroxid( 04M Phosphate buffer 05 ml 02M Phosphate

(pH 76)

Bicarbonate buffered Os04 Final concentrations

40 Osmium tetroxide 05 ml 20 25 Sodium bicarbonate 05 ml 125

(0625 gm2S ml water)

Adjust the pH of the bicarbonate buffer to 72 with HCl just before mixing with the Os04 The pH will rise to about 74 when it IS

diluted with Os04

The period of secondary fixation should be between ~ and 1 hour at room temperature depending on the size of the pieces of tissue Os04 penetrates much more slowly than glutaraldehyde If large (thicker than 1 mm) pieces have been used for primary fixation it would be advisable to cut them up before placing them in the secondary fixative Dehydrate and embed the tissues as usual

3

STEVE TORRENCES FIXATIVE

For marine embryos and larval tissues (especially ascidians)

A Primary fixative

approx osmolality

_______ 2 0 Glutaraldehyde 200

zfgtC 02M -shySodium cacodylate 375

0275M Sucrose 300

1 0 Tannic acid 85

960

B Preparation of fixative

Combine the following according to recommendation in section D

r80 Glutaraldehyde 100 ml J-ff 2 ~

Isj()O8M Sodium cacodylate 100 ml ~+--

055M Sucrose 200 ml

1annic acid powder 040 gm

cent(56- - 0--- tit Y17 r--shyC Stock solution information

c S -teth-Sodium cacodylate MW = 214

08 x 214 = 1712 1712 gm + 100 ml water 08M solution

[ Sucrose MW 3423 )

055 x 3423 = 1882 1882 gm + 100 ml water 0S5M solution

D Combining reagents

Tannic acid breaks down slowly in the light The powder needs to be protected from exposure to light It should be added to the fixative just before use Adjust the pH to 74 with 02N HCl after all components (including the tannic acid) have been combined The final osmolality should be about 960

4

E

F

( ~

G

H

Primary fixation methodology

Pre-cool the fixative and fix for 2-3 hours on ice Rinse the specimens briefly with a small amount of the fixative at the beginning or fix in a large volume of fixative since otherwise the calcium in the sea water may cause the tannic acid to precipitate

Buffer rinse

Rinse the tissues 3 or 4 times in the following solution to ensure removal of excess tannic acid Tannic acid precipitates with osmium tetroxide This solution should also be cooled on lce

1- 08M Sodium cacodylate 1 part 21M0fCtS-t(0 ~I

055M Sucrose 2 part (037H) I=- ~~x

The final concentrations are listed ln parentheses 1 M 1- ( Jl C

I~

p f J M j r C rD$( 01 ltgt

OSlt~ ( 61 ~ Second~y- ~ative J (N

40 Osmium tetroxide 1 part (2) 25 Sodium bicarbonate 1 part 025) ~

Adjust the pH of the bicarbonate buffer to 72 with Hel just before mixing it with Os04 Pre-cool the fixative on lce Fix for 1-2 hours on lce It may be necessary to rinse the specimens once with this fixative at the beginning to remove the last of the tannic acid if a brown precipitate forms I rr rl(t IMc Jo tL

IT vG-4te-J ( f~e-If c JDehydration infiltration and embedding

L -rtJ 6 J La-fS (1Ltdc1t r~cy ) Dehydration should be carried out on ice through the 90 step nit j

specimens should be brought to room temperature in the first chan ~ e of absolute solvent The best results are obtained when acetone or isopropanol are used as dehydrating agents and as antemedia

If ethanol is used as a dehydrating agent it must be followed by propylene oxide as an antemedium

Dr Steven Torrence took Zoology 583 several years ago He investigated the fine structure of some post-larval ascidians that attach to solid surfaces such as rocks wood and plastic Petri dishes He initially fixed detached specimens in bicarbonate buffered osmium tetroxide in combination with the standard Epon method The results were acc e ptable but not outstanding Later he modified his technique by omitting propylene oxide so that he could fix animals that were attached to plastic Petri dishes (Propylene oxide dissolves these dishes) Even though he used the same exact techniques repeatedly his results were extremely poor He could not obtain results that were even close to those obtained in his initial studies

5

c) Salt solution (034M)

~odium chlorid MW 5845 034M x 5845 r 9--87 -gmliter

397 gm200 ml

Add water to make 1 liter or 200 ml

It is convenient to have available a 1OM solution of sodium chloride (5845 gmliter) if you wish to modify the o~molality of the basic fixitive It can be diluted to the appropriate strength and used in place of the 034M solution recommended in the basic formula ~en an 8 solution of glutaraldehyde is used it is necessary to use a stock solution of 0747M sodium chloride in order to end up with the appropriate final concentrations of the three components

c Preparation of fixative from stock solutions using 25 glutaraldehyde

250 Glutaraldehyde 5 ml 034M Sodium chloride 20 ml 04M Phosphate bu f fe r 25 ml

50 ml

Preparation of fixative from stock solutions using 8 glutaraldehyde

80 Glutaraldehyde 10 ml 075M Sodium chloride 6 ml O4r Phospbate bu f fe r 16 ml

32 ml

The final solution of fixative should have an osmolality of 970 if all of the solutions have been properly prepared and mixed in the correct proportions It is advisable to check the osmolality with an osmometer The osmolality of seawater at the Friday Harbor Laboratories is usually about~but varies with the season ~

Fdmary poundxoo prp Vi 1J ( 0 d d 0D

The glutaraldehyde must penetrate the tissue before it can react with and stabilize the organic compounds in the cells and intercellular substances Small organisms (lmm in thickness) can be placed directly in the fixative Sometimes larger organisms or pieces of tissue can be adequately fixed without reducing their size but it is a good practice to fix pieces of several different sizes (See note under secondary fixation)

2

Fix the tissue for about 1 hour at room temperature Longer and shorter times can be tried Some specimens seem to be undamaged after as long as several days in the fixative Some small specimens (eggs protozoans) can be fixed in 15 minutes or less After primary fixation in glutaraldehyde the tissue may be rinsed in a phosphate buffer solution for 10-15 minutes or alternatively the tissue can be transferred directly into a small amount of the secondary fixative (see below) for a few seconds to dilute out the glutaraldehyde solution

Phosphate Buffer Rinsing Solution (optional)

Mix equal parts of 04M phosphate buffer stock solution and 06M sodium chloride

Final concentrations

02M Phosphate buffer pH 76 420 moms 03M Sodium chloride 560 moms

E Secondary fixation

Secondary fixation in Os04 is necessary to stabilize the lipids and improve the contrast for electron microicopy Either phosphate or bicarbonate buffered Os04 can be used I (RAC) prefer bicarbonate as a buffer for ascidians and mollusks

Phosphate buffered Os04 Final concentrations

40 Osmium tetroxide U5 ml Osmium tetroxid( 04M Phosphate buffer 05 ml 02M Phosphate

(pH 76)

Bicarbonate buffered Os04 Final concentrations

40 Osmium tetroxide 05 ml 20 25 Sodium bicarbonate 05 ml 125

(0625 gm2S ml water)

Adjust the pH of the bicarbonate buffer to 72 with HCl just before mixing with the Os04 The pH will rise to about 74 when it IS

diluted with Os04

The period of secondary fixation should be between ~ and 1 hour at room temperature depending on the size of the pieces of tissue Os04 penetrates much more slowly than glutaraldehyde If large (thicker than 1 mm) pieces have been used for primary fixation it would be advisable to cut them up before placing them in the secondary fixative Dehydrate and embed the tissues as usual

3

STEVE TORRENCES FIXATIVE

For marine embryos and larval tissues (especially ascidians)

A Primary fixative

approx osmolality

_______ 2 0 Glutaraldehyde 200

zfgtC 02M -shySodium cacodylate 375

0275M Sucrose 300

1 0 Tannic acid 85

960

B Preparation of fixative

Combine the following according to recommendation in section D

r80 Glutaraldehyde 100 ml J-ff 2 ~

Isj()O8M Sodium cacodylate 100 ml ~+--

055M Sucrose 200 ml

1annic acid powder 040 gm

cent(56- - 0--- tit Y17 r--shyC Stock solution information

c S -teth-Sodium cacodylate MW = 214

08 x 214 = 1712 1712 gm + 100 ml water 08M solution

[ Sucrose MW 3423 )

055 x 3423 = 1882 1882 gm + 100 ml water 0S5M solution

D Combining reagents

Tannic acid breaks down slowly in the light The powder needs to be protected from exposure to light It should be added to the fixative just before use Adjust the pH to 74 with 02N HCl after all components (including the tannic acid) have been combined The final osmolality should be about 960

4

E

F

( ~

G

H

Primary fixation methodology

Pre-cool the fixative and fix for 2-3 hours on ice Rinse the specimens briefly with a small amount of the fixative at the beginning or fix in a large volume of fixative since otherwise the calcium in the sea water may cause the tannic acid to precipitate

Buffer rinse

Rinse the tissues 3 or 4 times in the following solution to ensure removal of excess tannic acid Tannic acid precipitates with osmium tetroxide This solution should also be cooled on lce

1- 08M Sodium cacodylate 1 part 21M0fCtS-t(0 ~I

055M Sucrose 2 part (037H) I=- ~~x

The final concentrations are listed ln parentheses 1 M 1- ( Jl C

I~

p f J M j r C rD$( 01 ltgt

OSlt~ ( 61 ~ Second~y- ~ative J (N

40 Osmium tetroxide 1 part (2) 25 Sodium bicarbonate 1 part 025) ~

Adjust the pH of the bicarbonate buffer to 72 with Hel just before mixing it with Os04 Pre-cool the fixative on lce Fix for 1-2 hours on lce It may be necessary to rinse the specimens once with this fixative at the beginning to remove the last of the tannic acid if a brown precipitate forms I rr rl(t IMc Jo tL

IT vG-4te-J ( f~e-If c JDehydration infiltration and embedding

L -rtJ 6 J La-fS (1Ltdc1t r~cy ) Dehydration should be carried out on ice through the 90 step nit j

specimens should be brought to room temperature in the first chan ~ e of absolute solvent The best results are obtained when acetone or isopropanol are used as dehydrating agents and as antemedia

If ethanol is used as a dehydrating agent it must be followed by propylene oxide as an antemedium

Dr Steven Torrence took Zoology 583 several years ago He investigated the fine structure of some post-larval ascidians that attach to solid surfaces such as rocks wood and plastic Petri dishes He initially fixed detached specimens in bicarbonate buffered osmium tetroxide in combination with the standard Epon method The results were acc e ptable but not outstanding Later he modified his technique by omitting propylene oxide so that he could fix animals that were attached to plastic Petri dishes (Propylene oxide dissolves these dishes) Even though he used the same exact techniques repeatedly his results were extremely poor He could not obtain results that were even close to those obtained in his initial studies

5

Fix the tissue for about 1 hour at room temperature Longer and shorter times can be tried Some specimens seem to be undamaged after as long as several days in the fixative Some small specimens (eggs protozoans) can be fixed in 15 minutes or less After primary fixation in glutaraldehyde the tissue may be rinsed in a phosphate buffer solution for 10-15 minutes or alternatively the tissue can be transferred directly into a small amount of the secondary fixative (see below) for a few seconds to dilute out the glutaraldehyde solution

Phosphate Buffer Rinsing Solution (optional)

Mix equal parts of 04M phosphate buffer stock solution and 06M sodium chloride

Final concentrations

02M Phosphate buffer pH 76 420 moms 03M Sodium chloride 560 moms

E Secondary fixation

Secondary fixation in Os04 is necessary to stabilize the lipids and improve the contrast for electron microicopy Either phosphate or bicarbonate buffered Os04 can be used I (RAC) prefer bicarbonate as a buffer for ascidians and mollusks

Phosphate buffered Os04 Final concentrations

40 Osmium tetroxide U5 ml Osmium tetroxid( 04M Phosphate buffer 05 ml 02M Phosphate

(pH 76)

Bicarbonate buffered Os04 Final concentrations

40 Osmium tetroxide 05 ml 20 25 Sodium bicarbonate 05 ml 125

(0625 gm2S ml water)

Adjust the pH of the bicarbonate buffer to 72 with HCl just before mixing with the Os04 The pH will rise to about 74 when it IS

diluted with Os04

The period of secondary fixation should be between ~ and 1 hour at room temperature depending on the size of the pieces of tissue Os04 penetrates much more slowly than glutaraldehyde If large (thicker than 1 mm) pieces have been used for primary fixation it would be advisable to cut them up before placing them in the secondary fixative Dehydrate and embed the tissues as usual

3

STEVE TORRENCES FIXATIVE

For marine embryos and larval tissues (especially ascidians)

A Primary fixative

approx osmolality

_______ 2 0 Glutaraldehyde 200

zfgtC 02M -shySodium cacodylate 375

0275M Sucrose 300

1 0 Tannic acid 85

960

B Preparation of fixative

Combine the following according to recommendation in section D

r80 Glutaraldehyde 100 ml J-ff 2 ~

Isj()O8M Sodium cacodylate 100 ml ~+--

055M Sucrose 200 ml

1annic acid powder 040 gm

cent(56- - 0--- tit Y17 r--shyC Stock solution information

c S -teth-Sodium cacodylate MW = 214

08 x 214 = 1712 1712 gm + 100 ml water 08M solution

[ Sucrose MW 3423 )

055 x 3423 = 1882 1882 gm + 100 ml water 0S5M solution

D Combining reagents

Tannic acid breaks down slowly in the light The powder needs to be protected from exposure to light It should be added to the fixative just before use Adjust the pH to 74 with 02N HCl after all components (including the tannic acid) have been combined The final osmolality should be about 960

4

E

F

( ~

G

H

Primary fixation methodology

Pre-cool the fixative and fix for 2-3 hours on ice Rinse the specimens briefly with a small amount of the fixative at the beginning or fix in a large volume of fixative since otherwise the calcium in the sea water may cause the tannic acid to precipitate

Buffer rinse

Rinse the tissues 3 or 4 times in the following solution to ensure removal of excess tannic acid Tannic acid precipitates with osmium tetroxide This solution should also be cooled on lce

1- 08M Sodium cacodylate 1 part 21M0fCtS-t(0 ~I

055M Sucrose 2 part (037H) I=- ~~x

The final concentrations are listed ln parentheses 1 M 1- ( Jl C

I~

p f J M j r C rD$( 01 ltgt

OSlt~ ( 61 ~ Second~y- ~ative J (N

40 Osmium tetroxide 1 part (2) 25 Sodium bicarbonate 1 part 025) ~

Adjust the pH of the bicarbonate buffer to 72 with Hel just before mixing it with Os04 Pre-cool the fixative on lce Fix for 1-2 hours on lce It may be necessary to rinse the specimens once with this fixative at the beginning to remove the last of the tannic acid if a brown precipitate forms I rr rl(t IMc Jo tL

IT vG-4te-J ( f~e-If c JDehydration infiltration and embedding

L -rtJ 6 J La-fS (1Ltdc1t r~cy ) Dehydration should be carried out on ice through the 90 step nit j

specimens should be brought to room temperature in the first chan ~ e of absolute solvent The best results are obtained when acetone or isopropanol are used as dehydrating agents and as antemedia

If ethanol is used as a dehydrating agent it must be followed by propylene oxide as an antemedium

Dr Steven Torrence took Zoology 583 several years ago He investigated the fine structure of some post-larval ascidians that attach to solid surfaces such as rocks wood and plastic Petri dishes He initially fixed detached specimens in bicarbonate buffered osmium tetroxide in combination with the standard Epon method The results were acc e ptable but not outstanding Later he modified his technique by omitting propylene oxide so that he could fix animals that were attached to plastic Petri dishes (Propylene oxide dissolves these dishes) Even though he used the same exact techniques repeatedly his results were extremely poor He could not obtain results that were even close to those obtained in his initial studies

5

STEVE TORRENCES FIXATIVE

For marine embryos and larval tissues (especially ascidians)

A Primary fixative

approx osmolality

_______ 2 0 Glutaraldehyde 200

zfgtC 02M -shySodium cacodylate 375

0275M Sucrose 300

1 0 Tannic acid 85

960

B Preparation of fixative

Combine the following according to recommendation in section D

r80 Glutaraldehyde 100 ml J-ff 2 ~

Isj()O8M Sodium cacodylate 100 ml ~+--

055M Sucrose 200 ml

1annic acid powder 040 gm

cent(56- - 0--- tit Y17 r--shyC Stock solution information

c S -teth-Sodium cacodylate MW = 214

08 x 214 = 1712 1712 gm + 100 ml water 08M solution

[ Sucrose MW 3423 )

055 x 3423 = 1882 1882 gm + 100 ml water 0S5M solution

D Combining reagents

Tannic acid breaks down slowly in the light The powder needs to be protected from exposure to light It should be added to the fixative just before use Adjust the pH to 74 with 02N HCl after all components (including the tannic acid) have been combined The final osmolality should be about 960

4

E

F

( ~

G

H

Primary fixation methodology

Pre-cool the fixative and fix for 2-3 hours on ice Rinse the specimens briefly with a small amount of the fixative at the beginning or fix in a large volume of fixative since otherwise the calcium in the sea water may cause the tannic acid to precipitate

Buffer rinse

Rinse the tissues 3 or 4 times in the following solution to ensure removal of excess tannic acid Tannic acid precipitates with osmium tetroxide This solution should also be cooled on lce

1- 08M Sodium cacodylate 1 part 21M0fCtS-t(0 ~I

055M Sucrose 2 part (037H) I=- ~~x

The final concentrations are listed ln parentheses 1 M 1- ( Jl C

I~

p f J M j r C rD$( 01 ltgt

OSlt~ ( 61 ~ Second~y- ~ative J (N

40 Osmium tetroxide 1 part (2) 25 Sodium bicarbonate 1 part 025) ~

Adjust the pH of the bicarbonate buffer to 72 with Hel just before mixing it with Os04 Pre-cool the fixative on lce Fix for 1-2 hours on lce It may be necessary to rinse the specimens once with this fixative at the beginning to remove the last of the tannic acid if a brown precipitate forms I rr rl(t IMc Jo tL

IT vG-4te-J ( f~e-If c JDehydration infiltration and embedding

L -rtJ 6 J La-fS (1Ltdc1t r~cy ) Dehydration should be carried out on ice through the 90 step nit j

specimens should be brought to room temperature in the first chan ~ e of absolute solvent The best results are obtained when acetone or isopropanol are used as dehydrating agents and as antemedia

If ethanol is used as a dehydrating agent it must be followed by propylene oxide as an antemedium

Dr Steven Torrence took Zoology 583 several years ago He investigated the fine structure of some post-larval ascidians that attach to solid surfaces such as rocks wood and plastic Petri dishes He initially fixed detached specimens in bicarbonate buffered osmium tetroxide in combination with the standard Epon method The results were acc e ptable but not outstanding Later he modified his technique by omitting propylene oxide so that he could fix animals that were attached to plastic Petri dishes (Propylene oxide dissolves these dishes) Even though he used the same exact techniques repeatedly his results were extremely poor He could not obtain results that were even close to those obtained in his initial studies

5

E

F

( ~

G

H

Primary fixation methodology

Pre-cool the fixative and fix for 2-3 hours on ice Rinse the specimens briefly with a small amount of the fixative at the beginning or fix in a large volume of fixative since otherwise the calcium in the sea water may cause the tannic acid to precipitate

Buffer rinse

Rinse the tissues 3 or 4 times in the following solution to ensure removal of excess tannic acid Tannic acid precipitates with osmium tetroxide This solution should also be cooled on lce

1- 08M Sodium cacodylate 1 part 21M0fCtS-t(0 ~I

055M Sucrose 2 part (037H) I=- ~~x

The final concentrations are listed ln parentheses 1 M 1- ( Jl C

I~

p f J M j r C rD$( 01 ltgt

OSlt~ ( 61 ~ Second~y- ~ative J (N

40 Osmium tetroxide 1 part (2) 25 Sodium bicarbonate 1 part 025) ~

Adjust the pH of the bicarbonate buffer to 72 with Hel just before mixing it with Os04 Pre-cool the fixative on lce Fix for 1-2 hours on lce It may be necessary to rinse the specimens once with this fixative at the beginning to remove the last of the tannic acid if a brown precipitate forms I rr rl(t IMc Jo tL

IT vG-4te-J ( f~e-If c JDehydration infiltration and embedding

L -rtJ 6 J La-fS (1Ltdc1t r~cy ) Dehydration should be carried out on ice through the 90 step nit j

specimens should be brought to room temperature in the first chan ~ e of absolute solvent The best results are obtained when acetone or isopropanol are used as dehydrating agents and as antemedia

If ethanol is used as a dehydrating agent it must be followed by propylene oxide as an antemedium

Dr Steven Torrence took Zoology 583 several years ago He investigated the fine structure of some post-larval ascidians that attach to solid surfaces such as rocks wood and plastic Petri dishes He initially fixed detached specimens in bicarbonate buffered osmium tetroxide in combination with the standard Epon method The results were acc e ptable but not outstanding Later he modified his technique by omitting propylene oxide so that he could fix animals that were attached to plastic Petri dishes (Propylene oxide dissolves these dishes) Even though he used the same exact techniques repeatedly his results were extremely poor He could not obtain results that were even close to those obtained in his initial studies

5

Dr Torrence eventually found that the combination of ethanol and Epon when used for infiltration causes extensive and serious demage to tissues The reasons for this are not understood and of course were unexpected He expended a considerable amount of time about a year trying new fixatives It was only after many months that the cause of the major problem finally emerged from a series of planned experiments In retrospect the experience may have been worthwhile because the neVI fixative he formulated is considerably better than anything that was previously available for solitary ascidian embryos larvae and post-larval tissues We can now also appreciate that primary and secondary fixations are not necessarily the only critical stages in tissue preparation that have an important influence on the quality of preservation of ultrastructural details

The acetone or isopropanol should be completely free of water for the final stages of dehydration

The dehydrating agents can be kept absolute by storing them with 4A molecular sieves (Sigma catalog number M-2635)

Infiltration may be carried out with diluted solutions of the dehydrating agent and Epon as 1n the standard procedures that involve the use of propylene oxide

Although Steve Torrence usually omitted propylene oxide there does not appear to be any loss of quality of preservation Vlhen propylene oxide is used in the sequence following acetone or isopropanol

The addition of tannic acid to glutaraldehyde fixatives has been found to enhance preservation of both membranes (Saito et al 1978 ~ Cell BioI 79 601) and microfiliments (Begg et al 1978 ~ Cell Biol~846) The success of this method for ascidians also depends on proper dehydration and infiltration

The results of many experiments are summarized In the following table by Dr Steven Torrence

6

Results of uSIng various fixatives and dehydrating gents in differunt combinations (From Dr Steven Torrence 1981)

Bicarbonateshy Dunlaps Torrences Os04 fixation fixation fixation

I DEHYDRATION I

Ethanol Isopropanol I X X

X X

X X

X Acetone X X X X X X

INFILTRATION Ethanol X X X Isopropanol X X Acetone X X X Propylene oxide X X X X X

QUALITY OF PRESERVATION

Molgula larvae f -2 F + + -2 +2 - - - +3 +3 +3 Boltenia larvae +2 +2 Ascidia larvae +3 Corella embryos +3

----- shy --- shy

Each vertical column represents a separate fixation and embedment

7

-------

BICARBONATE BUFFERED OSMIUM TETROXIDE

Wood R and J Luft J Ultrastruct Res 12 22-45 (1965)

Fixative

40 Os04 1 part (20)

25 Sodium bicarbonate 1 part (125) (pH 72)

Mix sodium bicarbonate with water and adjust pH with 1 N HCl just before use Precool the fixative on ice after mixing Fix small pieces for 1 hr

This 1S an excellent secondary fixative that is used with a variety of primary fixatives containing aldehydes It can be used alone as a fixative but it does not preserve microtubules T-tubules smooth endoplasmic reticulum or delicate arrays of micr6filaments in some cells

KARNOVSKYS FIXATIVE

Recommended for vertebrate and insect tissues Not recommended for marine invertebrates Reference N J Karnovsky J Cell BioI 27 l37A (1965) ~

4 ~ Clt (tx-shy

A Primary Fixative 11 0 5 ut il C ~jr-(- JL

80 Paraformaldehyde sol 250 ml

500 Glutaraldehyde 50 ml

02 M Cacodylate buffer 200 ml 16 0 (pH 74 - 76)

Anhydrous Calcium 250 mg chloride --- ( ~ofF4500 ml cz C) Cgt c) shy

-- 2 0 1 0 - (J CJ Th e final pH of the fixative is 72 The osmolality 1S sathid to re ( 3lt0 2 10 Drs Ben Trump and Ruth Bulger recommend dilutin~ e ~

x a t i ve wit h equa 1 par t s 0 f Iva t e r for m a mmali an kid n e y Th e formula may be modified by omitting calcium chloride and substituting 02 M phosphate buffer

Preparation of paraformaldehyde Dissolve 2 gm paraformaldehyde in 25 ml water by heating (in a hood) to 65 0 c and stirring Add one to three drops of 1 N NaOH with continuous stirring until the solution

8

clears~ A slight milkiness may persist Cool before mIxlng with other reagents

Processing

Fix at r oom temperature Large pIeces (3-4 mm) of tissue may be fixed and then cut into smaller pieces after half an hour Fix 2-5 hrs

Wash tissues In cold 01 M buffer for 3-12 hrs

B Secondary Fi~ative

Post fix in cold S-collidine buffered Os04

2-4 Osmium Tetroxide 2 parts

02 M S-collidine buffer (pH 74) 1 part

Dehydrate and embed as usual

9

• Richard Apdf
• Cloney-TEM-fixation-manual

Results of uSIng various fixatives and dehydrating gents in differunt combinations (From Dr Steven Torrence 1981)

Bicarbonateshy Dunlaps Torrences Os04 fixation fixation fixation

I DEHYDRATION I

Ethanol Isopropanol I X X

X X

X X

X Acetone X X X X X X

INFILTRATION Ethanol X X X Isopropanol X X Acetone X X X Propylene oxide X X X X X

QUALITY OF PRESERVATION

Molgula larvae f -2 F + + -2 +2 - - - +3 +3 +3 Boltenia larvae +2 +2 Ascidia larvae +3 Corella embryos +3

----- shy --- shy

Each vertical column represents a separate fixation and embedment

7

-------

BICARBONATE BUFFERED OSMIUM TETROXIDE

Wood R and J Luft J Ultrastruct Res 12 22-45 (1965)

Fixative

40 Os04 1 part (20)

25 Sodium bicarbonate 1 part (125) (pH 72)

Mix sodium bicarbonate with water and adjust pH with 1 N HCl just before use Precool the fixative on ice after mixing Fix small pieces for 1 hr

This 1S an excellent secondary fixative that is used with a variety of primary fixatives containing aldehydes It can be used alone as a fixative but it does not preserve microtubules T-tubules smooth endoplasmic reticulum or delicate arrays of micr6filaments in some cells

KARNOVSKYS FIXATIVE

Recommended for vertebrate and insect tissues Not recommended for marine invertebrates Reference N J Karnovsky J Cell BioI 27 l37A (1965) ~

4 ~ Clt (tx-shy

A Primary Fixative 11 0 5 ut il C ~jr-(- JL

80 Paraformaldehyde sol 250 ml

500 Glutaraldehyde 50 ml

02 M Cacodylate buffer 200 ml 16 0 (pH 74 - 76)

Anhydrous Calcium 250 mg chloride --- ( ~ofF4500 ml cz C) Cgt c) shy

-- 2 0 1 0 - (J CJ Th e final pH of the fixative is 72 The osmolality 1S sathid to re ( 3lt0 2 10 Drs Ben Trump and Ruth Bulger recommend dilutin~ e ~

x a t i ve wit h equa 1 par t s 0 f Iva t e r for m a mmali an kid n e y Th e formula may be modified by omitting calcium chloride and substituting 02 M phosphate buffer

Preparation of paraformaldehyde Dissolve 2 gm paraformaldehyde in 25 ml water by heating (in a hood) to 65 0 c and stirring Add one to three drops of 1 N NaOH with continuous stirring until the solution

8

clears~ A slight milkiness may persist Cool before mIxlng with other reagents

Processing

Fix at r oom temperature Large pIeces (3-4 mm) of tissue may be fixed and then cut into smaller pieces after half an hour Fix 2-5 hrs

Wash tissues In cold 01 M buffer for 3-12 hrs

B Secondary Fi~ative

Post fix in cold S-collidine buffered Os04

2-4 Osmium Tetroxide 2 parts

02 M S-collidine buffer (pH 74) 1 part

Dehydrate and embed as usual

9

• Richard Apdf
• Cloney-TEM-fixation-manual

-------

BICARBONATE BUFFERED OSMIUM TETROXIDE

Wood R and J Luft J Ultrastruct Res 12 22-45 (1965)

Fixative

40 Os04 1 part (20)

25 Sodium bicarbonate 1 part (125) (pH 72)

Mix sodium bicarbonate with water and adjust pH with 1 N HCl just before use Precool the fixative on ice after mixing Fix small pieces for 1 hr

This 1S an excellent secondary fixative that is used with a variety of primary fixatives containing aldehydes It can be used alone as a fixative but it does not preserve microtubules T-tubules smooth endoplasmic reticulum or delicate arrays of micr6filaments in some cells

KARNOVSKYS FIXATIVE

Recommended for vertebrate and insect tissues Not recommended for marine invertebrates Reference N J Karnovsky J Cell BioI 27 l37A (1965) ~

4 ~ Clt (tx-shy

A Primary Fixative 11 0 5 ut il C ~jr-(- JL

80 Paraformaldehyde sol 250 ml

500 Glutaraldehyde 50 ml

02 M Cacodylate buffer 200 ml 16 0 (pH 74 - 76)

Anhydrous Calcium 250 mg chloride --- ( ~ofF4500 ml cz C) Cgt c) shy

-- 2 0 1 0 - (J CJ Th e final pH of the fixative is 72 The osmolality 1S sathid to re ( 3lt0 2 10 Drs Ben Trump and Ruth Bulger recommend dilutin~ e ~

x a t i ve wit h equa 1 par t s 0 f Iva t e r for m a mmali an kid n e y Th e formula may be modified by omitting calcium chloride and substituting 02 M phosphate buffer

Preparation of paraformaldehyde Dissolve 2 gm paraformaldehyde in 25 ml water by heating (in a hood) to 65 0 c and stirring Add one to three drops of 1 N NaOH with continuous stirring until the solution

8

clears~ A slight milkiness may persist Cool before mIxlng with other reagents

Processing

Fix at r oom temperature Large pIeces (3-4 mm) of tissue may be fixed and then cut into smaller pieces after half an hour Fix 2-5 hrs

Wash tissues In cold 01 M buffer for 3-12 hrs

B Secondary Fi~ative

Post fix in cold S-collidine buffered Os04

2-4 Osmium Tetroxide 2 parts

02 M S-collidine buffer (pH 74) 1 part

Dehydrate and embed as usual

9

• Richard Apdf
• Cloney-TEM-fixation-manual

clears~ A slight milkiness may persist Cool before mIxlng with other reagents

Processing

Fix at r oom temperature Large pIeces (3-4 mm) of tissue may be fixed and then cut into smaller pieces after half an hour Fix 2-5 hrs

Wash tissues In cold 01 M buffer for 3-12 hrs

B Secondary Fi~ative

Post fix in cold S-collidine buffered Os04

2-4 Osmium Tetroxide 2 parts

02 M S-collidine buffer (pH 74) 1 part

Dehydrate and embed as usual

9

• Richard Apdf
• Cloney-TEM-fixation-manual