Immunohistochemistry

IHC Staining Protocol for FFPE slides

(A) Deparaffinize slides: Xylene (2x5 min), 100% EtOH (2x3 min), 95% EtOH (1 min), 80% EtOH (1 min), ddH₂O (≥ 5 min)

(B) Staining

1. Antigen retrieval (select one)
   1. EDTA buffer
      • 1 mM EDTA + 0.05% Tween 20 in DI water, pH 8.0
      • Immerse slides in EDTA buffer in slide container
      • Place slide container in a steamer for 30 minutes
      • Cool for 30 minutes in the same buffer
   2. Citrate buffer
      • 10 mM sodium citrate + 0.05% Tween 20 in DI water, pH 6.0
      • Immerse slides in citrate buffer in slide container
      • Place slide container in steamer for 30 minutes
      • Cool for 30 minutes in the same buffer
   3. Proteinase K
      • Apply Proteinase K solution for 30 s
      • Rinse slides
2. Block endogenous peroxidase activity: 200 mL MeOH + 4 mL 30% H₂O₂; incubate for 40 min at room temperature on shaker
3. Wash 2x5 minutes in PBS
4. Using PAP pen, draw around sections
5. Block the section for 1 hour in blocking buffer (5% fetal horse serum, 1 mg/mL BSA, 0.05% Tween 20, 1X PBS) in a humidified chamber at RT
6. Remove the blocking buffer
7. Add the primary antibody, diluted to the desired concentration (see table below) in blocking buffer. Incubate 1.5 hours at RT in humidified chamber (or overnight at 4°C in humidified chamber)
8. Remove the primary antibody
9. Wash slides 3x 3 minutes in 1x PBS
10. Add ImPRESS Reagents (Vector Labs) to species match the primary antibody. Incubate  sections for 30 minutes at room temperature in humidified chamber
11. Wash slides 3x3 minutes in 1x PBS
12. Add DAB (Vector, #SK4105) (1 drop/1 mL) and incubate at room temperature
    1. DAB is an extreme carcinogen, so use appropriate caution
    2. Incubation time has to be determined for each antibody. On the first incubation, watch development under a microscope; look for brown staining but don’t over-incubate
13. Stop the reaction by immersion in water
14. Counterstain with Hematoxylin QS (Vector, #H3404). Incubate for 30 seconds at room temperature
15. Stop the reaction by immersion in water

 (C) Dehydration and Cover

1. 95% EtOH (2 min), 100% EtOH (2x 3 min), Xylene (2x 5 min)
2. Cover slide while moist with Cytoseal #60 ; Lay slides at an angle to dry overnight.

 

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Primary Antibodies – Anti-Human

Antibody	Species	Retrieval	Dilution	DAB	Company	Catalog #
TIM-3	Rabbit	EDTA	1/400	2 min	Cell Sig.	45208
Cleaved Caspase 3	Rabbit	EDTA	1/200	2 min	Cell Sig.	9661
CD4	Mouse	EDTA Citrate	1/50	3 min	Thermo	MS-1528-S0
CD45	Mouse	EDTA Citrate	1/100	2 min	Thermo	MS-355-P0
CD206	Rabbit	EDTA	1/500	4.5 min	Sigma	HPA004114
DC-LAMP	Goat	EDTA	1/100	45 sec	R&D	AF4087
Galectin 9	Mouse	EDTA	1/100	45 sec	Novus	45619
Granzyme B	Rabbit	EDTA	1/200	4 min	Novus	100-684

 

Secondary Reagents

Antibody	Company	Catalog #
Anti-Mouse ImPRESS	Vector	MP7401
Anti-Rabbit ImPRESS	Vector	MP7402
Anti-Goat ImPRESS	Vector	MP7405

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