DAB Staining on Frozen Sections Protocol
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DAB staining on frozen sections Protocol Defrost the slide Fixed the tissue using PFA or Ethanol for 12 minutes Quenching the endogenous enzyme by using 3% H 2 O 2 for 20 minutes Wash with 1x PBS 3 times, 5 minutes each Blocking the section using 10% BSA blocking solution or 1.5% Normal Goat/Horse Serum for 20 minutes Dilute the primary antibody with the blocking solution and apply on the slide for overnight at 4 °C or at room temperature for 2 hours Wash with 1xPBS 3 times, 5 minutes each Apply 2 nd antibody on the slide (1 in 200), and incubate at room temperature for 1 hour Preparation of A+B Complex by add 5ul A and 5ul B in 1ml 1xPBS with 0.2% tween20, mix well and sit for 30 minutes before use Wash the slide with 1xPBS for 3 times, 5 minutes each, apply AB complex onto the slide and incubate at room temperature for 30m minutes Wash again to remove the uncoupled AB complex, stain with DAB staining solution DAB staining solution preparation: 5ml H 2 O 2 drops of Buffer stock solution 4 drops of DAB stock solution 2 drops of 30% H2O2 solution Mix well and apply on the slide
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DAB staining
Transcript of DAB Staining on Frozen Sections Protocol
DAB staining on frozen sections Protocol
Defrost the slide
Fixed the tissue using PFA or Ethanol for 12 minutes
Quenching the endogenous enzyme by using 3% H2O2 for 20 minutes
Wash with 1x PBS 3 times, 5 minutes each
Blocking the section using 10% BSA blocking solution or 1.5% Normal Goat/Horse Serum for 20 minutes
Dilute the primary antibody with the blocking solution and apply on the slide for overnight at 4 °C or at room temperature for 2 hours
Wash with 1xPBS 3 times, 5 minutes each
Apply 2nd antibody on the slide (1 in 200), and incubate at room temperature for 1 hour
Preparation of A+B Complex by add 5ul A and 5ul B in 1ml 1xPBS with 0.2% tween20, mix well and sit for 30 minutes before use
Wash the slide with 1xPBS for 3 times, 5 minutes each, apply AB complex onto the slide and incubate at room temperature for 30m minutes
Wash again to remove the uncoupled AB complex, stain with DAB staining solution
DAB staining solution preparation: 5ml H2O2 drops of Buffer stock solution4 drops of DAB stock solution2 drops of 30% H2O2 solutionMix well and apply on the slide
