I've been conducting DAB stains for nuclear proteins, but I am consistently seeing an artifact like blurriness, as shown in Image 1. It's been largely at random, with only some slides experiencing the same artifact, even within the same run of the protocol. It has occasionally affected only one slide, but sometimes it can affect most or all of them in a single run.
Image 1 comes from the negative control for Image 2. They come from the same tissue block and sectioning. The only difference between their protocols (as far as I am aware) is whether primary antibody was applied -- but clearly something else is occurring.
Does anyone have any ideas on what I might be doing wrong, or how I can adjust my protocol to avoid this artifact?
Comment if you want more images.
Protocol:
Deparaffinizine Tissue (3x4 min xylene, 2x3 min 100% EtOH, 1x3 min 95% EtOH, 1x3 min 70% EtOH, 1x3 min 50% EtOH, PBS)
Heated EDTA-Tris antigen retrieval buffer, then wash with PBS
3% H2O2 10 min, then wash with PBS
0.3% triton X-100 in PBS 10 min room temp, then wash with PBS
Sniper block 8 min, then wash with PBS
Incubate with Primary in 1% BSA-PBS overnight, then wash with PBS
Incubate with biotinylated secondary in 1% BSA-PBS for 1hr, then wash with PBS
Incubate with streptavidin link in 1% BSA-PBS for 1hr, then wash with PBS
Place in H2O, then stain with DAB
Wash with H2O, counterstain with hematoxylin, wash with H2O again, then dehydrate (2 x 3 dips in 50% EtOH, 2 x 30 sec in 70% EtOH, 1 x 1 min in 95% EtOH, 2 x 3 min in 100% EtOH, 2 x 3 min in Xylene)
Mount coverslip using Permount solution
hello. DAB is light sensitive it should be done in dark condition and slides should be kept in humid chamber. dab you are using may be too old.....The thoroughly washed sections should be stained with freshly prepared 3% 3,3'-diaminobenzidine tetrahydrochloride hydrate (DAB) solution for 20-30 min. DAB oxidizes in the presence of peroxidase and hydrogen peroxide resulting in the deposition of a dark brown precipitate at the site of enzymatic activity.....
When morphology of the tissue is affected during IHC, most times the HIER-step is the culprit. What is your technique for HIER. Are you sure, that all slides of a run are heated equally?
Household-Microwave-ovens have hotspots. Waterbaths may have a temperature-graduation (up-down). Number of slides can affect the necessary heating time.
I agree Gudrun Lang. I measure the temperature after I put the slides in the waterbath.
I agree with the rest of opinions. In addition, you can also check that the mounting process has been performed correctly. For example, some times if there is an excess of mounting medium the image may be blurred.
Are you the only one using the microscope where the images were captured?
- Badges
- Science method