TITLE: Immunohistochemistry/Immunofluorescence
AUTHORS: Michael Henderson

[DESCRIPTION]
This protocol details about the immunohistochemisty/immunofluorescence staining techniques for tissue.

[STEPS]
SECTION: Day 1
1. Label slides with antibody and treatment to be used.
SECTION: Day 1
2. De-paraffinize slides in fresh xylenes, then in a descending ethanol series.
SECTION: Day 1
3. Formic Acid Retrieval (If necessary, do here). 

SECTION: Day 1
4. Microwave antigen retrieval (CA; If necessary do here).
SECTION: Day 1
5. Immerse in freshly prepared Methanol/H2O2 (150 mLMethanol + 30 mL stock 30% H2O2) 30 min. 
 
Then, wash in running tap H2O for 10 min.
  



SECTION: Day 1
6. Wash in 0.1 Molarity (M)Tris buffer, pH 7.6 5 min. Discard all Tris washes.

SECTION: Day 1
7. Block in 0.1 Molarity (M)Tris/2% FBS (Tris/FBS) 5 min+. Keep blocking solution for up to 2 weeks @ 4 °C.

SECTION: Day 1
8. Dilute primary antibodies in Tris/FBS, and prepare humidified chamber(s) by soaking towel in the middle of the slide chamber(s).
SECTION: Day 1
9. Wipe excess fluid off back of slides and from around tissue and apply 200 µL of primary antibody to slides. Make sure antibodies cover all sections.

 

SECTION: Day 1
10. Incubate at 4 °C in humidified chamber 5 min.

SECTION: Day 1
2.1. De-paraffinize slides 5 minin fresh xylenes. (1/2)

SECTION: Day 1
2.2. De-paraffinize slides 5 minin fresh xylenes. (2/2)
SECTION: Day 1
2.4. De-paraffinize slides in ethanol 100% for 1 min.
SECTION: Day 1
2.5. De-paraffinize slides in ethanol 95% for 1 min.
SECTION: Day 1
2.6. De-paraffinize slides in ethanol 80% for 1 min.
SECTION: Day 1
2.7. De-paraffinize slides in ethanol 70% for 1 min.
SECTION: Day 1
3.1. Immerse slides in ddH2O for 1 min, place in recycled FA for 5 min  and wash in running tap H2O for 10 min.

SECTION: Day 1
4.1. Dilute antigen unmasking solution (Vector Labs, citric acid) 1:100 in dH2O (2.5 mL/250 mLdH2O/boat).

SECTION: Day 1
4.2.  Place in Biogenex EZ-Retriever microwave for 15 min at 95 °C.

SECTION: Day 1
4.3. Cool for 20 min at95 Room temperature. 

SECTION: Day 1
4.4. Wash slides for 10 min in running tap H2O.

SECTION: Day 2
11. Rinse off antibody from tissue using Tris.
 

SECTION: Day 2
12. Wash in Tris 5 min.

SECTION: Day 2
13. Block in Tris/FBS 5 min.

SECTION: Day 2
14. Dilute Vector biotinylated IgG 1:1000 in Tris/FBS and apply200 µL to wiped slides. 

SECTION: Day 2
15. Incubate at 4 Room temperature in humidified chamber 60 min.

SECTION: Day 2
16. Rinse biotinylated IgG using Tris.
SECTION: Day 2
17. Wash in Tris 5 min.

SECTION: Day 2
18. Block in Tris/FBS 5 min. 

SECTION: Day 2
19. Mix AB solution (Vector peroxidase standard) in Tris/FBS to a dilution of 1:000 (ie add 1 µLof A and 1 µL of B to 1 mL of 0.1 Molarity (M) Tris/2%FBS). Vortex and let sit 15 min before use. Then, apply 200 µL of AB solution to wiped slides.

SECTION: Day 2
20. Incubate at 4 Room temperature in humidified chamber for 60 min.

SECTION: Day 2
21. Rinse off AB using Tris.
SECTION: Day 2
22. Immerse in Tris 5 min.

SECTION: Day 2
23. Make Vector DAB solution (1 drop of DAB per mL of Stable DAB Buffer) .
SECTION: Day 2
24. Apply 200 µL of DAB to each slide and incubate until a visible brown signal is seen and well developed.
 

SECTION: Day 2
25. Rinse with Tris and place in dH2O. Wash 5 min in dH2O. Filter Harris hematoxylin.

SECTION: Day 2
26. Counterstain briefly with Harris hematoxylin (~15 s, depending on age).

SECTION: Day 2
27. Wash in running tap H2O 5 min.

SECTION: Day 2
28. Dehydrate and clear in ascending ethanol and xylenes. 
SECTION: Day 2
29. Coverslip with cytoseal.
SECTION: Day 2
30. Dry in tissue processor closet 5 min.

SECTION: Day 2
14.1. *For immunofluorescence, dilute fluorescent secondary antibodies 1:500 in Tris/FBS and apply 200 µL to wiped slides. Keep slides in the dark from here on. Incubate at 4 °C in humidified chamber overnight or at 4 Room temperaturefor 180 min or overnight at 4 °C . Proceed to Day 3.

SECTION: Day 2
28.1. Dehydrate and clear in 70% ethanol for 1 min  and 70% xylenes for  5 min.
SECTION: Day 2
28.2. Dehydrate and clear in 80% ethanol for 1 min  and 80% xylenes for  5 min.
SECTION: Day 2
28.3. Dehydrate and clear in  95% ethanol for 1 min  and 95% xylenes for  5 min.
SECTION: Day 2
28.4. Dehydrate and clear in 100% ethanol for 1 min  and 100%  xylenes for  5 min.
SECTION: Day 2
28.5. Dehydrate and clear in 100% ethanol for 1 min  and 100%  xylenes for  5 min.
SECTION: Day 3 (Immunofluorescence)
31. Filter Sudan Black. This process can take a long time, so start early.
SECTION: Day 3 (Immunofluorescence)
32. Rinse off AB using Tris.
SECTION: Day 3 (Immunofluorescence)
33. Wash in running tap H2O for 5 min.

SECTION: Day 3 (Immunofluorescence)
34. Wash in Tris for 5 min in green boats.

SECTION: Day 3 (Immunofluorescence)
35. Sudan Black Treatment (0.3% Sudan Black B in 70% Ethanol) 
SECTION: Day 3 (Immunofluorescence)
36. Wash in 0.1 Molarity (M) Tris 5 min in green boats.

SECTION: Day 3 (Immunofluorescence)
37. Coverslip using non-photobleaching reagent (FluorMount with DAPI). Allow to dry completely before imaging on scanner.
SECTION: Day 3 (Immunofluorescence)
35.1. Use a control slide (usually 1 positive primary and 1 secondary only) to titrate for background reduction without changing signal intensity (usually 10 s to 1 min).

SECTION: Day 3 (Immunofluorescence)
35.2.  Image before and after Sudan black treatment for various times. 
SECTION: Day 3 (Immunofluorescence)
35.3. Treat all slides identically.
SECTION: Day 1
2.3. De-paraffinize slides in ethanol 100% for 1 min.