Download - Tissue preparation (group 4)
TISSUE PREPARATION
1. RAFEAH RUSLI 03-200904-00277
2. RAFIDAH ABRAHAM 03-200904-00324
3. SAMSON AK CLEMENT 03-200904-00359
4. SANDRA LOUIS 03-200904- 00274
5. SARTIKA AMRAN 03-200904-00180
6. VERA DIANE 03-200904-00244
By the end presentation, we able to:
Identify the fixative used for immunohistochemistry.
Understand the sectioning for immunohistochemistry.
Described the whole mount preparation.
OBJECTIVE
CONTENT
Fixation
Sectioning
Whole mount preparation
Tissue preparation is the cornerstone of immunohistochemistry.
To ensure the preservation of tissue architecture and cell morphology, prompt and adequate fixation is essential.
Inappropiate or prolonged fixation may significantly diminish the antibody binding capability.
There is no one universal fixative that is ideal for the demonstration of all antigens.
FIXATION
Many antigens can be successfully demonstrated in formalin-fixed paraffin-embedded tissue sections.
The discover and development of antigen retrieval techniques further enhanced the use of formalin as routine fixative for immunohistochemistry in many research laboratories.
For best results, vertebrate tissues (especially neuronal tissues) usually require fixation by transcardial perfusion for optimal tissue preservation
The most common fixatives used for immunohistochemistry are the followings:- 4% paraformaldehyde in 0.1M phosphate buffer.- 2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate buffer.- PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2% lysine in 0.1M phosphate buffer.- 4% paraformaldehyde with 0.05% glutaraldehyde
(TEM immunohistochemistry).
Some antigens will not survive even moderate amounts of aldehyde fixation.
Tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat without infiltrating with sucrose.
The sections should be kept frozen at -20 C or lower until fixation with cold acetone or alcohol.
The sections can be processed using standard immunohistochemical staining protocols.
Paraffin wax has remained the most widely used embedding
medium for diagnostic histopathology in routine histological
laboratories. The largest proportion of material for immunohistochemistry
is formalin-fixed, paraffin-embedded. Paraffin sections produce satisfactory results for the
demonstration of majority of tissue antigens with the use of
antigen retrieval techniques.
SECTIONING
Certain cell antigens do not survive routine fixation and
paraffin embedding. The disadvantage of frozen sections includes:
i) Poor morphology
Ii) Poor resolution at higher magnifications
Iii) Special storage needed
Iv) Limited retrospective studies and cutting difficulty over
paraffin sections.
VIBRATOMEA vibratome is an instrument that is similar
to a microtome but uses a vibrating razor
blade to cut through tissue.
The vibration amplitude, the speed, and
the angle of the blade can all be controlled.
Fixed or fresh tissue pieces are embedded
in low gelling temperature agarose.(Some
have had success without using the agarose
to embed.)
The resulting agarose block containing the
tissue piece is then glued to a metal block
and sectioned while submerged ina a water
or buffer bath.
Individual sections are then collected with
a fine brush and transferred to slides or
multiwell plates for staining.
Advantages of vibratome :
No need to dehydrate tissues prior to embedding, thus decreased loss of cell constituents.
No messy paraffin embedding. No need to deparaffinise and rehydrate sections prior to
immunostaining. No high temperatures or harsh chemical treatments that
may lead to antigen instability. No special microtome blades required. Less chance of artifacts caused by parrafin embedding or
freezing. Increased tissue autofluoresecence due to paraffin
embedding being avoided.
Less wait period from tissue sampling to time of immunolabelling.
The tissue is not processed through organic solvents or high
heat, which can destroy the antigenicity when
immunocytochemistry. The morphology of tissue sections is not disrupted due to no
freezing and thawing needed. Vibratome sections are often used for floating
immunostaining, especially for pre-embedding EM
immunohistochemistry.
The disadvantage of vibratome sections :
Instead of ribbons, single sections are cut and collected which are more delicate and difficult to handle.
Sections are generally thicker than those obtained with paraffin methods.
penetration of antibodies and other reagents may be slower and thus longer incubation times may be necessary.
thick sections may be difficult to image with the microscope. Securing vibratome sections to glass slides can be difficult or
impossible, due to the thickness of the sections. the sectioning process is slow and difficult with soft and
poorly fixed tissues. The chatter marks or vibratome lines are often appeared in
the sections.
Small blocks of tissue (less than 5 mm thick) can be processed as whole mounts.
The advantage of WMP :The results provide three dimensional information about the location of antigens without the need for reconstruction from sections.
Whole Mount Preparation
However, the major limitation of using whole mounts is antibody penetration may not be complete in the tissue, resulting in uneven staining or false negative staining.
Treatment : Triton X-100 or saponin are used routinely for whole mount immunohistochemistry to enhance penetration of the antibody.
MICROSCOPE OPTIC
Day 1: Dissection and Fixation of Mammary Glands 1. Dissect out mammary gland and spread on glass
slide. 2. Fix tissue O/N in Carnoy's fixative at RT.
Day 2: Rehydration and Staining of Glands 1. Wash in 70% EtOH 2 x 15 min. 2. Wash in 50% EtOH 2 x 10 min. 3. Wash in 30% EtOH 2 x 10 min. 4. Wash in 10% EtOH 2 x 10 min. 5. Wash in distilled H2O 1 x 5 min. 6. Stain O/N in carmine alum stain
Mammary Gland Whole Mount Preparations
Day 3: Clearing and Mounting of Glands 1. Wash in 70% EtOH 2 x 15 min. 2. Wash in 95% EtOH 2 x 15 min. 3. Wash in 100% EtOH 2 x 15 min. 4. Clear in xylene approximately 30 minutes
(or until fat is sufficiently cleared from glands).
5. Mount with - 1mL SecureMount and glass cover slip.
Carnoy's Fix: 6 parts 100% EtOH 3 parts CHCl3 1 part glacial acetic acid
Carmine Alum Stain: Place 1g carmine (Sigma C1022) and 2.5g
aluminum potassium sulfate (Sigma A7167) in 500mL dH20 and boil 20 min. Adjust final vol. to 500mL with H20. Filter and add thymol crystal as preservative. Refrigerate. Can be used for several months. Discard when color becomes weak.
There is no standard tissue preparation schedule for the optimal demonstration of all antigens.
Factors involved in all aspects of tissue preparation can affect immunoreactivity, so it is important that precise details of the preparation schedule are given when reporting immunocytochemical studies, rather than using the general term "routinely fixed and processed“.
CONCLUSION
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