DAB Staining of Fixed Mouse Brain Tissue Sections

Blocking and primary antibody incubation.

Pull all sections into 1x dPBS in 24-well netwells.

Wash 3 times with 1x dPBS, 10 mins each.

Wash 3 times with 1x TBS, 10 mins each.

Quench endogenous peroxidase activity with quenching buffer, 5 mins at RT. Just covering the sections is sufficient, and drain excess buffer by dabbing on kimwipes before and after.

Wash 3 times with 1x TBS, 10 mins each.

Block in TBS++++ for 75 mins at RT on rocker, 2 mL/well.

Incubate in primary antibody diluted in TBS++++ on rocker at RT O/N, 500 µL/well in a new 24-well plate without netwell insert.

Secondary antibody incubation and DAB development

Wash 3 times with 1x TBS, 5 mins each in netwell insert.

Wash 2 times with Tris, 5 mins each.

Wash 2 times with TBS, 5 mins each.

Wash 2 times with PBS, 10 mins each.

Sections can be stored in PBS at 4C.

Mount onto Superfrost Plus slides labeled with pencil and allow to dry completely.

Incubate in secondary antibody at a 1:300 dilution in TBS++++ for 2 hrs, 500 µL/well in a 24-well plate without netwell insert.

15 mins prior to end of incubation, prepare ABC complex:

• Both buffer A and B are 1:300 dilution in TBS++++
• Incubate in RT beaker of water for 30 mins, in drawer

Wash 3 times with 1x TBS, 5 mins each in netwell insert.

Incubate 2 hours in ABC complex in TBS++++ at RT on rocker without netwell insert.

Wash 3 times with 0.1 M Tris buffer, pH 8.0, 5 mins each in netwell insert.

Prepare 1x DAB solution:

• Thaw aliquots of 50x DAB, 1 tube is around 200 µL
• Add 50x DAB into 0.1M Tris buffer
• Vortex for 5 sec to mix well, sit in dark for 10 mins
• Right before development, add 30% H2O2 (1:10000 dilution), vortex to mix well
• Distribute 2 mL to each well on a 12-well plate

Prepare an additional 12-well plate with Tris buffer to start washes immediately after development.

Develop DAB reaction, minimizing time difference between wells. Drain excess buffer by dabbing on kimwipes before and after.

Dehydration and clearing.

Wash 2 x 2min in ddH2O.

Wash 2 x 2min in 70% EtOH.

Wash 2 x 2min in 95% EtOH.

Wash 2 x 2min in 100% EtOH.

Wash 2 x 2min in Xylene.

Coverslip using Permount mounting medium.

Let dry in fume hood overnight then store in drawer in slide box until imaging.