Table 2: Steps of Iba1 immunostaining on paraffin sections by using “vectastain ABC kit (Cat No PK-4001).”

StepsTime of incubationTemperature

Deparaffinize slides on a heat block
Deparaffinize with Xylene5 mins * 2 timesRT
100% ethyl alcohol5 minsRT
70% ethyl alcohol5 minsRT
Distilled water5 minsRT
PBS 1x5 minsRT
Antigen retrieval: Antigen Unmasking solution (Vector Laboratories; H3300) (9.375 mL of antigen unmasking solution in 1 liter of distilled water)15 mins95°C
Blocking (Vector laboratories rabbit kit) (15 µL/1 mL of PBS)60 mins37°C
Anti-Iba1 antibody (1 : 100 dilution)120 mins37°C
PBS 1x wash3 mins × 3 times
Secondary antibody (Vector laboratories rabbit kit) (5 µL/mL of PBS)60 mins37°C
PBS wash3 minsRT
ABC reagent (Vector laboratories rabbit kit) (1 µL Reagent A + 1 µL reagent B + 10 µL of PBS1x)30 mins37°C
DAB substrate (Vector laboratories rabbit kit) (500 µL distilled water + 100 µL buffer + 200 µL DAB + 100 µL H2O2)5–10 mins (till a brown colour develops)RT
Distilled water5 mins
Hematoxylin (Counter staining)30 sec
Running water wash
50% Ethanol10 sec/20 dips
70% Ethanol12 sec/24 dips
100% Ethanol14 sec/28 dips
Xylene3 mins × 3 times
Mounting with refrax mounting medium and observed under light microscope

RT: room temperature.