Deparaffinize and rehydrate

• Melt paraffin from sections at 55°C for 15 minutes (optional if slides are not freshly cut).
• Immerse slides 2x in Xylene, 2x in 100% EtOH, 1x 95% EtOH, 1x 70% EtOH, 1x 50%, 2x deionized (di) water, 5 minutes each solution.

Retrieve

• Perform antigen retrieval if required with antibody of interest. If not required go to next step.
• To do this place slides in Abcam Antigen Retrieval Buffer (10mM citrate pH = 6.0) and heat retrieve.
• Rinse 1x for 1 minute in di water, 2x 3min in Tris buffered saline pH = 7.6 + 0.1% tween 20.

Quench endogenous peroxidases

• Incubate sections in Bloxall for 10 minutes at room temperature (alternatively use freshly diluted 10% H₂O₂ in 1x PBS or include 10% H₂O₂ in MeOH during the deparaffinization steps).
• Rinse slides in di water for 1 minute.
• Equilibrate slides 5 minutes in buffer.

M.O.M. Block

• Prepare M.O.M. Mouse IgG Blocking Reagent (Vector Labs, cat#MP-2400) by combining 2 drops (~90 µl) of M.O.M. Blocking Reagent stock solution to 2.5 ml of 1XPBS.
• Wipe excess buffer from slides with kimwipe or gauze avoiding tissue.
• Cover sections in M.O.M. block solution and incubate 60 minutes RT.
• Wash for 2 x 2 minutes in 1XPBS.

Serum block

• Wipe slides.
• Cover sections in 2.5% normal serum 20 minutes RT (species should match the source of the secondary antibody that will be used).
• Do not rinse, just drain off excess block.

Antibody Incubation

• Apply diluted primary antibody (concentration provided with website image should be initially tested along with one-fold dilution stronger and weaker).
• Wash 1x 5 min in Buffer at RT

ImmPRESS Incubation

• Apply ready to use ImmPRESS Vector antibody to species of interest to sections fr 30 minutes at RT.
• Wash 2x 5 minutes in Buffer at RT.

Detection in DAB

• Make ImmPact DAB solution following manufacturer′s instructions. Mix well.
• Cover sections with solution and develop while observing under a brightfield microscope.
• Stop reaction in water rinse once desired color intensity is attained (usually 30 seconds- 2 minutes).
• Wash 5 minutes in water.

Counterstain

• Cover sections in OPTIK Hematoxylin for 60 seconds. Rinse well in tap water.
• Dip slides in clarifier for 30 seconds to remove excess hematoxylin. Rinse well in tap water.
• Dip slides in bluing solution for one minute and rinse in tap water to intensify blue color of counterstain.
• Run slides back through graded ethanols to xylenes. Three xylene stations are recommended.

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