Whenever I perform an in situ hybridization, black specks appear throughout the tissue within a few hours post mounting with glycerol gel. THey are not present while developing in B4 solution, and they are not present immediately after mounting. I purchased new NBT/BCIP, new glycerol gel, and kept them out of light but they still appear when I observe them under the scope. I also cleaned the objective of the microscope, but the specks are clearly within the tissue.
Glycerol Gel mountant ! For NBT/BCIP in situ use mount from Biomedia or Vectamount or Immunomount from Vector Laboratories. The same companies also offer organic counterstains which are compatible with these mounting media (e.g.,Vector Methyl Green, Vector Nuclear Fast Red).
I have to give it a try. Thank you! Has this happened to you as well?
As your NBT/BCIP are new then its very likely to be micro air bubbles, Always keep your mountant stored in a squeezy plastic dropper bottle and inverted. this way all those tiny air bubbles are at the bottom of the solution. Keeping it inverted apply in a steady one stroke line down the coverliide only when you have finished then stop squeezing the bottle, As soon as the pressure is off, the bottle will draw in a small amount of air, trcap your bottle and return it inveryed to your rack. Invert your slide onto the coverslip that way you can put sufficient pressure on the back of the slide to eliminate any further air pockets, If you place the coverslide ontop of the microscope slide. you can't then exert enough pressure to eliminate tiny bubbles without the coverslide sliding sideways & taking your section with it ! Good luck
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