A DAB immunohistochemistry for free-floating sections

Angie Chiang,February 2018

Reagents Needed
1XTBS
Triton X-100

30% H2O2(stored in 4º C fridge)

Normal Goat Serum(Most commercial serums should be ok. We use EMD Millipore, catalog # S26-LITER)

88% Formic Acid

Permount

Rabbit anti-human Aβ antibody (Thermo Fisher/Zymed,catalog # 71-5800)

Vectastain Elite ABC Kit, Rabbit IgG (Vector Laboratories, catalog # PK-6101)

DAB (Sigma; SIGMAFAST™ 3,3′-Diaminobenzidine tabletscatalog # D4418)

Ethanol (70%, 95% and 100%)

Xylene

Notes

• Use Netwell insertsto hold sections for all steps except antibody incubation (Corning, 24 mm/74 um, 6/plate, catalog # 3479). Use a #0 or #1 paint brush to move sections between plates and nets.

• All steps done at room temperature unless noted.

• All TBS washes done in Netwell inserts placed into Netwell reagent trays (Corning, catalog # 3517)

Procedure

Day 1:

Remove sections from cryoprotectant solution and wash 3 x 10 minutes in 1X TBS.

Move sections into 88% Formic Acidfor1 minute in nets.

Wash sections 3 x 10 minutes in 1X TBS.

Block exogenous peroxidase activity using 0.9% H2O2 in 0.01% TBST (TBS + 0.01% Triton X-100) for 30 min.

Wash sections 3 x 10 minutes in 1X TBS.

Block in 5% Normal Goat Serum in 0.3% TBST for 1 hour. Rock or rotate gently during this step.

Incubate in primary antibody diluted 1:500 in blocking solution overnight at 4º C with continuous gentle rocking or rotation. Move sections from Netwell inserts into 12 or 24 well tissue culture plates for this step.

Day 2:

Return sections from tissue culture plate to Netwells for wash: 3 x 10 min in 1X TBS.

Incubate in biotinylated secondary antibody from Vector kit diluted 1:500 in blocking solution for 1-2 hr. Move sections from Netwell inserts into 12 or 24 well tissue culture plates for this step. Be sure to use the serum and secondary antibody provided in the Vector kit.

Prepare the Vector ABC solution: add 2 drops Vector Solution A (equivalent to 100 ul) + 2 drops (100 ul) Solution B into5 ml of 1X TBS. Stand at RT for 30 min before use.

Wash sections 3 x 10 min in 1X TBS.

Incubate sections in ABC reagent for 90 min. Move sections from Netwell inserts into 12 or 24 well tissue culture plates for this step.

Return sections from tissue culture plate to Netwells for wash: 3 x 10 min in 1X TBS.

Prepare DAB solution: dissolve each tablet set into 15 ml deionized water. Be sure to use both tablets provided in the kit. Solution can be used immediately after tablets are dissolved.

Develop in DAB - use white Netwell reagent trays for this step (Corning, catalog # 3519)

Wash sections 3 x 10 min in 1X TBS.

Mount sections on Superfrost Plus slides. Air-dry overnight at room temperature.

Day 3:

Rehydrate slides in 1X TBS buffer. Use glass staining dishes for these steps.

Dehydrate slides through an alcohol series: 2 X 70% ethanol, 2 X 95% ethanol, 3 X 100% ethanol (2 minor 5 dunks in each)

Finish dehydration in 100% ethanol and move slides to xylene.

Coverslip with Permount and let slides dry overnight.