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IHC staining for pannexin-1 

1. Deparaffinization & hydration

Xylene for 5min x 3 times (I, II, III)

100% ethanol for 1min x 3 times (I, II, III)

95% ethanol for 1min;70% ethanol for 1min;

DI water for 5min x 3.

2. Antigen Retrieval with Vector Antigen Unmasking Solutions (partly adopted from DAKO AR protocol) 

Bring to boil waterbath. Prepare Antigen Retrieval (AR) solution by diluting a stock 1:106.67 in DI water(150μL/16mL). Bring to boil AR solution. Place slides into boiling working AR solution for 30min. Allow to cool, in AR buffer, for 30min.Rinse well with DI water.

3. Staining with BLOXALL® Endogenous Enzyme Blocking Solution (Vector) for 10 min RT. 

Rinse in TBST or PBS – 5min x 3

Cycle the section with PAP-pen

Avidin Application – 15 minRinse in TBST briefly Biotin Application – 15 min

Rinse in TBST – 11min x 3

Protein Block–20-60 min at RT or overnight at +4 ̊C.

Primary rabbit anti-PANX1 (human) Antibody 1:20-1:50 (from Sigma or Alomone) –overnight at 4 ̊C.

Rinse in TBST - 10min x 3Bionatylated goat anti-rabbit IgG secondary antibody, 1:150-1:200 (7.5-10μg/mL) (Vector) for 30-60 min at RT. 

Rinse in TBST – 10min x 3

Streptavidin-HRP (ready to use solution [1μg/mL]) – 30minRinse in TBST – 10min x 34. 

4. DAB application

Add 1 drop (~30μL) of the ImmPACT DAB reagent 1 to 1ml ImmPACT DAB diluent (bothfrom Vector), mix well (stable within 14 days at 2-8⁰C and 5 days at RT). Incubate for 2-10min. Wash for 5 min in water.

5. Counterstaining with hematoxylin

Hematoxylin (ready-to-use) –1-5 min.

Rinse with running tap water until rinse water is colorless.

Differentiate by dipping slides 10 times in acid rinse solution (2ml glacial acetic acid plus 98 deionized or distilled H2O).

Dipping 10 times in tap water. 

Bluing with solution (0.3mL NH4OH 30% stock to 100mLof tap water) by dipping 3-5 times. 

Rinse briefly (10-29 dips) in tap water.

Alternative: Hematoxylin QS – No bluing step!6. DehydrationReagent alcohol, 95% - 1-2min x 2

Absolute ethanol – 1-2min x 2

Xylene - 1-2min x 2 

Rapid Dehydration by Vector protocol: 99% isopropanol – at least 1 min x 2(twice)1. Permanent (non-aqueous) mounting with VectaMount® Express mounting media(H-5700) - 2 drops and coverglass. 

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