How to Test New Antibodies for How to Test New Antibodies for ImmunohistochemistryImmunohistochemistry

Finding a good antibodyFinding a good antibody

Provide control slidesProvide control slides

Pre-treatmentsPre-treatments

Detection methodsDetection methods

Fixation methodsFixation methods

Finding a good antibodyFinding a good antibodyBmi1 M R&D, Bmi1 R Cell, Bmi1M Milli, Bmi1 R Bethy

,

Finding a good antibodyFinding a good antibody Search the Web for commercially available Search the Web for commercially available

antibodies that have been tested by the vendor antibodies that have been tested by the vendor or reported in the literature to work on paraffin or reported in the literature to work on paraffin and/or frozen sections.and/or frozen sections.

Check the species of the immunogen for this Check the species of the immunogen for this antibody.antibody.

Consult the company to find out if it has been Consult the company to find out if it has been tested for IHC. tested for IHC.

Comparison betweenComparison betweenPolyclonal and monoclonal Polyclonal and monoclonal

Polyclonal AbPolyclonal Ab Monoclonal Ab Monoclonal AbInexpensive Inexpensive Expensive Expensivenon-specific non-specific specific specificbatch-to-batch batch-to-batch Recognizes multiple Recognizes one epitopeRecognizes multiple Recognizes one epitope

epitopeepitopeBatch-to-batch No or low batch-to- Batch-to-batch No or low batch-to-

variability batch variabilityvariability batch variability

Finding a good antibodyFinding a good antibodySuggested Ab companySuggested Ab company

Provide Provide control slidescontrol slides

Thromb R

(Thrombocyte)

Provide control slidesProvide control slides

Positive controlsPositive controls Providing the slides will most likely express Providing the slides will most likely express

the antigen. The best controls may not the antigen. The best controls may not necessarily be the tissue of interest but should necessarily be the tissue of interest but should be optimal for examining the staining be optimal for examining the staining conditions for the antibody.conditions for the antibody.

Tissue,age and species.Tissue,age and species.

Provide control slidesProvide control slides

Negative controlsNegative controls

knock-out tissueknock-out tissue

Tissue which does not express the antigenTissue which does not express the antigen

A blocking peptideA blocking peptide

Pre-treatments Pre-treatments

No pretreatmentNo pretreatment Heat antigen retrievalHeat antigen retrieval

PCBE, EDTA, BorgPCBE, EDTA, Borg Proteinase K or TrypsinProteinase K or Trypsin

Pre-treatments Pre-treatments

Detection methodsDetection methods Indirect fluorescent staining: Alexa 488, Alexa Indirect fluorescent staining: Alexa 488, Alexa

568568 Amplified fluorescent staining: ImmPF or Amplified fluorescent staining: ImmPF or

ImmP/RedImmP/Red HRP (horseradish peroxidase) staining: HRP (horseradish peroxidase) staining:

ImmPHRP or ABCHRPImmPHRP or ABCHRP

Fixation MethodsFixation Methods

Frozen section without fixationFrozen section without fixation 2% PF,4%PF or 10% buffered formalin fix 2% PF,4%PF or 10% buffered formalin fix

overnightovernight 4%PF fix 2 days4%PF fix 2 days

How to submit New Ab for IHCHow to submit New Ab for IHC Fill out IHC form.Fill out IHC form. Provide us with all possible antibody Provide us with all possible antibody

information and staining conditions from the information and staining conditions from the company and from the literature. company and from the literature.

Submit 4 positive control slides for testing Submit 4 positive control slides for testing with a new antibody.with a new antibody.

Fill out IHC form for new AbFill out IHC form for new Ab

1.1. Add a new record on the IHC formAdd a new record on the IHC form

2.2. Go to Layout #1 and fill in all fields on Go to Layout #1 and fill in all fields on part1part1

3.3. Go back to layout #6 Go back to layout #6

4.4. Close file and change the date (next Close file and change the date (next Monday) on the file nameMonday) on the file name

AntibodyAntibody SpeciesSpecies OrganizeOrganize StorageStorage ResourceResource ConcentrationConcentration Good for IHCGood for IHC Catalog#Catalog# CloneClone Exp.DateExp.Date ImmunogenImmunogen InformationSheetInformationSheet Cross ReactivityCross Reactivity PretreatmentPretreatment Target TissueTarget Tissue Target CellsTarget Cells Cytoplasma or Cytoplasma or

Nuclear stainingNuclear staining

How to test the New AbHow to test the New AbFirst test:First test:

Pre-treatment: No treatment and PCBEPre-treatment: No treatment and PCBE

Detection method: Alexa 488 or ImmPFDetection method: Alexa 488 or ImmPF

Second test:Second test:

Increase or decrease concentration.Increase or decrease concentration.

Change methodChange method

Third test:Third test:

Change pretreatment: PK , EDTA or BorgChange pretreatment: PK , EDTA or Borg

Last test: Frozen section or change fixation on Last test: Frozen section or change fixation on

paraffin sectionparaffin section

Interpretation of IHC resultsInterpretation of IHC results

Positive staining in right expression cells, Positive staining in right expression cells, Nuclear or cytoplasmic staining,Nuclear or cytoplasmic staining, Negative tissue no staining.Negative tissue no staining.

Interpretation of IHC resultsInterpretation of IHC results

1.Positive cells show staining but negative tissue 1.Positive cells show staining but negative tissue show light staining show light staining

Lower Ab concentration Lower Ab concentration

2. Very light staining in positive cells, no 2. Very light staining in positive cells, no

staining in negative tissue staining in negative tissue

Increase Ab concentration,Increase Ab concentration,

Repeat the staining,Repeat the staining,

Incubate 1Incubate 1stst Ab 2 days Ab 2 days

Update IHC resultsUpdate IHC results

Update Ab results on your IHC form with Update Ab results on your IHC form with good, no result and use ? for not sure.good, no result and use ? for not sure.

You need to send 1-2 photos of the new Ab to You need to send 1-2 photos of the new Ab to the Core for the database update before you the Core for the database update before you submit more IHC for this Ab. submit more IHC for this Ab.

Send photos with PowerPoint and write the Ab Send photos with PowerPoint and write the Ab expression information underneath the photo. expression information underneath the photo.

How to submit double IHCHow to submit double IHC

Look for the each primary Ab from which Look for the each primary Ab from which animal species. animal species.

Look for the pretreatment method.Look for the pretreatment method. Which Ab firstWhich Ab first Which color for which AbWhich color for which Ab

Animal speciesAnimal species

The animal species of each Ab should be different The animal species of each Ab should be different for double IHC. for double IHC.

We can work out two primary Abs from rabbit and We can work out two primary Abs from rabbit and use Alexa 488 and 568 method.use Alexa 488 and 568 method.

The ImmuPress method can change to Alexa 488 The ImmuPress method can change to Alexa 488 by increase primary Ab’s concentration by increase primary Ab’s concentration

5-10 times.5-10 times.

Pretreat methodPretreat method

Both primary Abs are same treatment.Both primary Abs are same treatment. PCBE and No: Do No first with HRP then PCBE and No: Do No first with HRP then

PCBE.PCBE. PK and No: Try No with PK or do Do No first PK and No: Try No with PK or do Do No first PCBE and PK Try PCBE with PK PCBE and PK Try PCBE with PK

Not work with PCBE only and PK only. Not work with PCBE only and PK only.

Which Ab to use as first Which Ab to use as first primary Abprimary Ab

1.1. Primary Ab from goatPrimary Ab from goat

2.2. GFP, RFPGFP, RFP

3.3. Strong IHC stainingStrong IHC staining

4.4. No treatment No treatment

Which color for which AbWhich color for which Ab

Blue: Use for the Ab with strong stainingBlue: Use for the Ab with strong staining Red: Use for the Ab with the staining on cell Red: Use for the Ab with the staining on cell

membrane or few area will be stained or lessmembrane or few area will be stained or less

background staining.background staining. Green: Use for the Ab with weak staining.Green: Use for the Ab with weak staining.