1. How to Test New Antibodies for Immunohistochemistry Finding a good antibody Provide control slides Pre-treatments Detection methods Fixation methods

2. Finding a good antibody Bmi1 M R&D, Bmi1 R Cell, Bmi1M Milli, Bmi1 R Bethy,

3. Finding a good antibody Search the Web for commercially available antibodies that have been tested by the vendor or reported in the literature to work on paraffin and/or frozen sections. Search the Web for commercially available antibodies that have been tested by the vendor or reported in the literature to work on paraffin and/or frozen sections. Check the species of the immunogen for this antibody. Check the species of the immunogen for this antibody. Consult the company to find out if it has been tested for IHC. Consult the company to find out if it has been tested for IHC.

4. Comparison between Polyclonal and monoclonal Polyclonal Ab Monoclonal Ab Polyclonal Ab Monoclonal Ab Inexpensive Expensive Inexpensive Expensive non-specific specific non-specific specific batch-to-batch batch-to-batch Recognizes multiple Recognizes one epitope Recognizes multiple Recognizes one epitope epitope epitope Batch-to-batch No or low batch-to- Batch-to-batch No or low batch-to- variability batch variability variability batch variability

5. Finding a good antibody Suggested Ab company

6. Provide control slides Thromb R (Thrombocyte)

7. Provide control slides Positive controls Positive controls Providing the slides will most likely express the antigen. The best controls may not necessarily be the tissue of interest but should be optimal for examining the staining conditions for the antibody. Providing the slides will most likely express the antigen. The best controls may not necessarily be the tissue of interest but should be optimal for examining the staining conditions for the antibody. Tissue,age and species. Tissue,age and species.

8. Provide control slides Negative controls knock-out tissue Tissue which does not express the antigen A blocking peptide

9. Pre-treatments No pretreatment No pretreatment Heat antigen retrieval Heat antigen retrieval PCBE, EDTA, Borg PCBE, EDTA, Borg Proteinase K or Trypsin Proteinase K or Trypsin

10. Pre-treatments

11. Detection methods Indirect fluorescent staining: Alexa 488, Alexa 568 Indirect fluorescent staining: Alexa 488, Alexa 568 Amplified fluorescent staining: ImmPF or ImmP/Red Amplified fluorescent staining: ImmPF or ImmP/Red HRP (horseradish peroxidase) staining: ImmPHRP or ABCHRP HRP (horseradish peroxidase) staining: ImmPHRP or ABCHRP

12. Fixation Methods Frozen section without fixation Frozen section without fixation 2% PF,4%PF or 10% buffered formalin fix 2% PF,4%PF or 10% buffered formalin fix overnight overnight 4%PF fix 2 days 4%PF fix 2 days

13. How to submit New Ab for IHC Fill out IHC form. Fill out IHC form. Provide us with all possible antibody information and staining conditions from the company and from the literature. Provide us with all possible antibody information and staining conditions from the company and from the literature. Submit 4 positive control slides for testing with a new antibody. Submit 4 positive control slides for testing with a new antibody.

14. Fill out IHC form for new Ab 1. Add a new record on the IHC form 2. Go to Layout #1 and fill in all fields on part1 3. Go back to layout #6 4. Close file and change the date (next Monday) on the file name

15. AntibodySpecies AntibodySpecies OrganizeStorage OrganizeStorage ResourceConcentration ResourceConcentration Good for IHCCatalog# Good for IHCCatalog# CloneExp.Date CloneExp.Date ImmunogenInformationSheet ImmunogenInformationSheet Cross ReactivityPretreatment Cross ReactivityPretreatment Target Tissue Target Tissue Target Cells Target Cells Cytoplasma or Cytoplasma or Nuclear staining Nuclear staining

16. How to test the New Ab First test: Pre-treatment: No treatment and PCBE Pre-treatment: No treatment and PCBE Detection method: Alexa 488 or ImmPF Detection method: Alexa 488 or ImmPF Second test: Increase or decrease concentration. Increase or decrease concentration. Change method Change method Third test: Change pretreatment: PK, EDTA or Borg Change pretreatment: PK, EDTA or Borg Last test: Frozen section or change fixation on paraffin section paraffin section

17. Interpretation 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.

18. Interpretation of IHC results 1.Positive cells show staining but negative tissue show light staining Lower Ab concentration 2. Very light staining in positive cells, no staining in negative tissue Increase Ab concentration, Repeat the staining, Incubate 1 st Ab 2 days

19. Update IHC results Update Ab results on your IHC form with good, no result and use ? for not sure. Update Ab results on your IHC form with good, no result and use ? for not sure. You need to send 1-2 photos of the new Ab to the Core for the database update before you submit more IHC for this Ab. You need to send 1-2 photos of the new Ab to the Core for the database update before you submit more IHC for this Ab. Send photos with PowerPoint and write the Ab expression information underneath the photo. Send photos with PowerPoint and write the Ab expression information underneath the photo.

21. How to submit double IHC Look for the each primary Ab from which animal species. Look for the each primary Ab from which animal species. Look for the pretreatment method. Look for the pretreatment method. Which Ab first Which Ab first Which color for which Ab Which color for which Ab

22. Animal species The animal species of each Ab should be different for double IHC. The animal species of each Ab should be different for double IHC. We can work out two primary Abs from rabbit and use Alexa 488 and 568 method. We can work out two primary Abs from rabbit and use Alexa 488 and 568 method. The ImmuPress method can change to Alexa 488 by increase primary Ab’s concentration The ImmuPress method can change to Alexa 488 by increase primary Ab’s concentration 5-10 times. 5-10 times.

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

24. Which Ab to use as first primary Ab 1. Primary Ab from goat 2. GFP, RFP 3. Strong IHC staining 4. No treatment

25. Which color for which Ab Blue: Use for the Ab with strong staining Blue: Use for the Ab with strong staining Red: Use for the Ab with the staining on cell membrane or few area will be stained or less Red: Use for the Ab with the staining on cell membrane 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.