Immunohistochemistry (IHC) of DRG

 Immunohistochemistry of mouse dorsal root ganglia.

1.     Euthanize mouse and dissect dorsal root ganglia into ice-cold Phosphate-Buffered Saline (PBS) pH 7.4.

2.     Transfer ganglia into 15 mL ice-cold fixative (4% paraformaldehyde in PBS) per sample.

3.     Post-fix tissues on ice for 1 hour.

4.     Wash tissues into filter-sterilized 30% sucrose in PBS: Place tissue into a 6 well plate filled with 30% sucrose, then carefully transfer with forceps from well to well. Perform at least 3 transfers.

5.     Cryo-protect tissues in 15 mL 30% sucrose overnight at 4°C, or until tissue sinks to bottom of tube.

6.     Rinse tissues into tissue freezing medium (Tissue-Tek OCT or similar) using a 6 well plate, same as with sucrose rinse step above.

7.     Freeze down tissues into a biopsy cryomold in bath of 95% ethanol and dry ice. Cryomolds can be stored in air-tight bags for up to 3 months at -80°C.

8.     Section tissues at -16°C into 14 µm sections. Temperature may need to be adjusted depending on individual cryostat. Proceed with staining. Alternatively, sections can be stored protected from air for up to 3 months at -80°C.

9.     Outline tissue sections 2-4 times with hydrophobic barrier pen (PAP pen or similar). Let barrier dry completely (10-30 minutes) at room temperature.

10.  Wash tissue with PBST (PBS pH 7.4 with 0.3% Triton X-100) 3 times for 5 minutes each. Washes should be performed directly on slides with gentle pipetting and vacuum aspiration.

11.  Incubate tissues in blocking solution 1-2 hours at room temperature using 200-700 µL blocking solution per slide in a humidified box. An overnight block can instead be performed at 4°C. Block solution consists of PBS with 0.1% Triton X-100 with 2.5% Horse Serum and 2.5% (w/v) cold ethanol fraction Bovine Serum Albumin. Make block fresh each day. Do not wash blocking solution prior to addition of primary antibody.

12.  Dilute desired primary antibody mixes at 1:1000 (or adjust concentration as needed) in Antibody Solution (PBS + 0.1% Triton X-100 with 0.5% Horse Serum and 0.5% cold ethanol fraction Bovine Serum albumin (w/v)). Incubate slides with 200-500 µL diluted primary antibody in Antibody Solution overnight at 4°C in humidified chamber. For no primary control, use Antibody Solution without added antibody. Antibody concentration may need to be adjusted based on antibody used.

13.  Wash slides 3 times with PBS 10 minutes each at room temperature.

14.  Prepare secondary antibody mixes at 1:1000 in Antibody Solution. Apply to appropriate slides same as with primary antibody step. Incubate 1-2 hours at room temperature in light-protected, humidified box.

15.  Wash slides 3 times with PBS 10 minutes each at room temperature.

16.  Mount slides in ~75 µL Fluoromount-G + DAPI or similar wet mounting medium and carefully apply no. 1.5 thickness coverslips. Wick away excess mounting medium from edges of coverslip and check for bubbles. Nail polish edges to seal. Do not use “quick dry” nail polish. Allow nail polish to dry completely in the dark before imaging. Image immediately or store slides in dark at 4°C up to one week.

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