Research Article
Cytomorphological and Cytochemical Identification of Microglia
Table 2
Steps of Iba1 immunostaining on paraffin sections by using “vectastain ABC kit (Cat No PK-4001).”
| | Steps | Time of incubation | Temperature |
| | Deparaffinize slides on a heat block | | | | Deparaffinize with Xylene | 5 mins * 2 times | RT | | 100% ethyl alcohol | 5 mins | RT | | 70% ethyl alcohol | 5 mins | RT | | Distilled water | 5 mins | RT | | PBS 1x | 5 mins | RT | | Antigen retrieval: Antigen Unmasking solution (Vector Laboratories; H3300) (9.375 mL of antigen unmasking solution in 1 liter of distilled water) | 15 mins | 95°C | | Blocking (Vector laboratories rabbit kit) (15 µL/1 mL of PBS) | 60 mins | 37°C | | Anti-Iba1 antibody (1 : 100 dilution) | 120 mins | 37°C | | PBS 1x wash | | 3 mins × 3 times | | Secondary antibody (Vector laboratories rabbit kit) (5 µL/mL of PBS) | 60 mins | 37°C | | PBS wash | 3 mins | RT | | ABC reagent (Vector laboratories rabbit kit) (1 µL Reagent A + 1 µL reagent B + 10 µL of PBS1x) | 30 mins | 37°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 water | 5 mins | | | Hematoxylin (Counter staining) | 30 sec | | | Running water wash | | | | 50% Ethanol | 10 sec/20 dips | | | 70% Ethanol | 12 sec/24 dips | | | 100% Ethanol | 14 sec/28 dips | | | Xylene | 3 mins × 3 times | | | Mounting with refrax mounting medium and observed under light microscope | | |
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RT: room temperature.
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