v Immunohistochemistry with APAAPstaining Authors S. Raffegerst Date 30-10-2007 Background Version 1.0 The immunohistochemistry method allows the identification of cells with expression of specific surface markers in tissue. Furthermore, this method allows the assessment of the distribution and localization of specific cells in their surroundings and their relationship to other cells. Additionally, this method offers the possibility to observe the localization of specific molecules in the cells themselves. This special protocol was developed for immunohistological staining of tissue-infiltrating lymphocytes, but it is also useful for other cell markers. The following antibodies are used for lymphocyte identification in tissue: CD3 (clone UCHT-1), CD4 (clone MT310) and CD8 (clone C8/144B) (Ebelt et al. 2007). MATERIALS REAGENTS: - cryosections of 5 µm thickness on SuperFrost Plus microscope slides (Menzel-Gläser, Cat.No.: J1800AMNZ) - Phosphate buffered saline (PBS) (Gibco by Invitrogen, Cat.No.: 14190-094) - acetone (Merck, Cat.No.: 100013) - DakoPen delimiting pen (Dako, Cat.No.: S2002) - albumin, bovine (Sigma, Cat.No.: A-9647) - human sera, type AB, male (plasma-derived) (Cambrex, Cat.No.: 14-491E) Helmholtz Zentrum Munich CCG Immune Monitoring Protocol: Immunohistochemistry with APAAP-staining 1
- N,N-dimethylformamide (Sigma, D-4254) - tetramisole hydrocloride (Sigma, L9756-5G) - naphtol AS phosphate disodium salt (Sigma, N 9252-5G) - sodium nitrite (Merck, Cat.No.: 106549) - distilled and filtered water, non-sterile (Millipore) - Mayers hemalum solution (Merck, Cat.No.: 109249) - Immumount (Thermo Electron Corporation, Cat.No.: 9990402) - aqua ad iniectabilia (B. Braun, Melsungen) - polyclonal rabbit anti-mouse immunoglobulins (DakoCytomation, Cat.No.: Z 0259) - APAAP, mouse monoclonal (DakoCytomation, Cat.No.: D0651) - specific antibodies - HCl solution (2mol/l, 2N) (Merck, Cat.No.: 109063) - New fuchsin (Merck, Cat.No.: 105226) - Folded filters (Whatman GmbH, Cat.No.: 10311647) EQUIPMENT: - optical light microscope - microtome for preparation of tissue cryosections - sterile Eppendorf tubes (1.5 ml or 2 ml) - crystal cuvettes for histology - crystal cuvettes for hemalaum bath and water baths - wet chamber for incubation of microscope slides - exhaust pump for washing the slides REAGENT SETUP: - prepare New fuchsin stock solution: dissolve 5 g Neufuchsin in 100 ml 2 N HCl solution - for antibody staining, specific antibodies, polyclonal rabbit anti-mouse and APAAP staining prepare 8% human serum solution with PBS - prepare specific antibody solutions with 8% human serum Helmholtz Zentrum Munich CCG Immune Monitoring Protocol: Immunohistochemistry with APAAP-staining 2
- prepare polyclonal rabbit anti-mouse immunoglobulin (1:20) dilution with 8% human serum solution for both incubation steps - prepare APAAP dilution (1:40) in 8% human serum solution for both incubation steps - prepare the AP and developing buffer for the developing solution - AP-buffer for developing solution: dissolve 12.1 g Tris-hydroxy(methyl-)aminomethan (0.1 M) and 5.84 g Sodium chloride in 1 l aqua ad injectabilia (water for injection) - developing buffer for developing solution dissolve 1.21g Tris-hydroxy(methyl-)aminomethan (0.1 M) and 5.85 g Sodium chloride in 1 l aqua ad injectabilia (water for injection) Tissue sections DISPOSITIONS FOR IMMUNOHISTOLOGICAL STAINING 1. prepare the 5 µm thick tissue cryosections with the microtome and place them on the SuperFrost Plus microscope slides 2. Let the sections dry for two days 3. After drying follow the next steps of APAAP staining or freeze the cryosections at -20 C or -80 C for storage APAAP Staining 1. at the time of immunohistological staining: thaw the microscope slides with the cryosections and let them dry again for 1 2 h and then follow the next steps 2. fill enough acetone in a crystal cuvette for histology and let the 100% acetone cool on ice until it is ice-cold (acetone can be reused up to 5 times) Helmholtz Zentrum Munich CCG Immune Monitoring Protocol: Immunohistochemistry with APAAP-staining 3
3. fixation: place the slides in the ice-cold acetone bath incubate for 20-30 min in the fixation bath 4. air drying: after the fixation bath, allow the slides to air dry until the acetone has evaporated 5. encircle the sections on the microscope slides with a liquid blocker (DakoPen) the circle around the tissue sections avoids leakage of the fluids during the incubation steps 6. now transfer the slides into a humid chamber and wet the tissue sections with PBS Attention: from this step onwards do not allow the tissue sections to dry until the staining has been completed 7. wash the section three times with PBS 8. blocking: pipette approximately 100 200 µl 2 % BSA/PBS solution onto the tissue sections incubate for 10 min at room temperature 9. wash three times with PBS 10. pipette 80 200 µl of specific antibody solution onto the sections incubate for 1 hour at room temperature 11. wash three times with PBS 12. pipette 80 200 µl of polyclonal rabbit anti-mouse (1:20 in 8% human serum) solution to the sections incubate for 30 minutes at room temperature 13. wash three times with PBS 14. pipette 80 200 µl of APAAP (1:40 in 8% human serum) solution onto the sections Helmholtz Zentrum Munich CCG Immune Monitoring Protocol: Immunohistochemistry with APAAP-staining 4
incubate for 30 minutes at room temperature 15. wash three times with PBS 16. pipette 80 200 µl of polyclonal rabbit anti-mouse (1:20 in 8% human serum) solution onto the sections incubate for 15 minutes at room temperature 17. now begin to prepare the developing solution and start with weighing the chemicals in different Ependorf tubes Substance amount in milligrams 1. tetramisole hydrocloride 30 mg 2. naphtol AS phosphate disodium salt 37.5 mg 3. sodium nitrite 30 mg 18. after the 15 minute incubation time, wash the slides again three times with PBS 19. pipette 80 200 µl of APAAP (1:40 in 8% human serum) solution on the sections incubate for 15 min at room temperature 20. wash the tissue section three times with PBS 21. after washing leave the slides covered with PBS, they should not be allowed to dry out 22. now prepare the developing solution Helmholtz Zentrum Munich CCG Immune Monitoring Protocol: Immunohistochemistry with APAAP-staining 5
Warning: toxic substances: work in a chemical exhaust hood during the developing time and with the developing solution 23. use a 100 ml beaker (or larger) and place it on a magnetic mixer 24. prepare the developing solution Solution Place of preparation substance volume A beaker tetramisole hydrocloride 30 mg AP-buffer 18.75 ml Developing-buffer 52.5 ml B Eppendorf tube sodium nitrite 30 mg New fuchsin stock solution 150 µl aqua ad iniectabilia 375 µl C Eppendorf tube naphtol AS phosphate 37.5 mg disodium salt dimethylformamide 450 µl 25. pipette solution B and then C into solution A Warning: the order of pipetting is important first B then C into A 26. let the solution mix for approximately 5 minutes 27. filter (with folded filters) the solution into a crystal cuvette for histology and place the microscope slides into the solution incubate for 20 min in the dark and on a mixer 28. transfer the microscope slides shortly into pure distilled water and then perform 29. nuclear counter staining in the Mayers hemalum solution for approximately 30 seconds Helmholtz Zentrum Munich CCG Immune Monitoring Protocol: Immunohistochemistry with APAAP-staining 6
30. quickly transfer the slides into a water bath with tap water and under flowing water allow the tissue to become blue approximately 10 minutes under flowing water 31. transfer the slides into a distilled water bath to stop the blueing reaction, without time limit (only some seconds are needed but they can stay in the water for longer) 32. the slides should remain wet until they are sealed 33. sealing: give one drop of Immu-Mount onto the tissue section, place a cover glass over the drop and smooth the Immu-Mount without forming bubbles on the tissue slide or damaging the tissue Analysis - with help of optical light microscopy the tissue can be analysed - positive cells are stained red Helmholtz Zentrum Munich CCG Immune Monitoring Protocol: Immunohistochemistry with APAAP-staining 7