IHC staining done -Heat retrieval-Blocking -Primary Ab overnight-Secondary ab -ABC kit-DAB- dehydration(Xylene 5 mins ) and mount. I loose lots of sectionns During the xylene (dehydration)step. What could I do to prevent this? Help!!!
Hi. If your sections are surviving antigen retrieval and other harsh methods, I am wondering why it cannot survive dehydration steps. Do you coat your slides with something before taking the sections onto them? For how long do you perform the dehydration step? Are your sections paraffin embedded or cryo? If it is paraffin, I hope "dehydration" means taking the sections from 50% ethanol to 100% ethanol and then xylene.
I do agree with Anupa, it is unusual. Did you use charge slides? Can you specify what type of tissue did you use? Some times if you use joint tissue including decalcified bone you may notice that part of tissue comes out from the slides.
We use slides coated with poly-l-lysine and sometimes double or triple coat. I also know a few people facing problems with decalcified condyle not sticking to slides. For some of them,it worked with VECTABOND. You may try that.
You can use a different chromogen like AEC. You might need to change your mounting media( AEC needs aquous mounting media. But you don't need to have Xylene steps. After hematoxylin staining. You just need to do tapwater wash(2-4 minutes) and dih2o wash(1 minute) before coverslipping step.
Are you using Tween or Triton in your reagents? They are detergents and can help sections detach from the slides. I know this might sound daft but how are you putting the reagents on? I have seen people "washing " the slides and squirting the PBS/TBS/Water directly onto the sections and wondering why the jet washed sections come off!
I use Tween. I use a pipette to wash the slides. Add the wash soln gently with a pipette all over the slide then drain the fluid off by touching the edge of the slide on a paper towel.
Tween is more gentle than Triton. Both get used to make a section more "permeable" to the reagents. You dont always need to do that, it depends on your primary.. Tween also gets used as a surfactant in other IHC reagents, so that the reagents overcome surface tension and flow evenly across the section. I would guess it is down to the drying 37C 24-48 hours and then once dry a bake for 20 mins at 60C. But joint work is difficult to get stuck on to slides.. Try a 50:50 mix of xylene:abs alchoholstaining trough in between the last alcohol for dehydration and the 1st clearing xylen troughe Good luck...
If they are baked vetically the paraffin can run and clump over tissue. So the 1st dewax in xylene stage, should be extended in time to ensure that all the wax is removed... Otherwise you can prevent the aqueous reagent from getting in to any "waxy" spots on the section and you end with a negativly stained area.
In continuation to IHC I have one more question for you all. I normally block with the serum from the host of the secondary antibody. for eg. my secondry is Donkey anti Mouse..so I use normal donkey serum to block my sections. Should I use the same serum 3% in PBST to dilute my primary or is it ok for me to use 3% BSA? thanks
There is no hard and fast answer to including the diluted blocking serum as part of the primary diluent... It very much depends on your particular primary. I would not include it as standard, but I have had to include it for the occasional primary. I work on the KISS principle (keep it simple stupid) then if it doesnt work you can start complicating it... And yes bake them horizontally.