I am currently repeating the same procedures with slightly different methods, but every time, my brain tissue slices disappear from the slides within a few weeks of coverslipping them. The slides have perfect brain tissue outlines where there are greyish debris-like material (maybe?) at everywhere outside of the slices and where the ventricles were, but where the tissue was is crystal clear.
Here are the steps I take:
1. Perfusion with 4% PFA and transferal of brains into the same PFA solution
2. Transferal of brains to 30% sucrose for 3 days
3. Section tissue via microtome and store in 96 well plates with PB+Azide
4. Transfer select tissue to gelatinous mounting solution to help place tissue onto the top of polarized slides
5. Let tissue on slides dry for two days
6. Wash the slides
- For some slides, the process included the consecutive rinsing with higher percentages of alcohol and then with xylenes
- For others, the process only involved rinsing with deionized water for 2 minutes
7. Add 50 uL of the Vectashield DAPI mounting media onto the slides and add coverslips at an angle to prevent bubbles
- For some slides, hardening version of the mounting media was used
- For others, the non-hardening version of it was used and nail polish was added to the edge of coverslips to prevent sliding
Please help. I only have a few days to figure this out and none of my lab members have seen anything like this before and I would like to prevent this from happening.
It seems to be the case that your sections were washed away during the several rinsing steps. If you only need a counterstain with Dapi, mount your sections on Superfrost plus Slides. For mounting use 0,45% NACl solution with a small drop of fluid soape which you use for dish washing (Pril in Germany). After 2 -3 days air drying in the fridge (to prevent fading of any fluophores which were applicated before) cover the sections with a mounting medium which contains Dapi. After a immunohistochemica reaction with fluophores as a detection system you can process is it in the same way. Good luck!
Omit step 5. as you are using vectashield mounting medium which itself is an water based mounting media so you dont need to dry your tissues in air for 2 days or with alcohol or xylene. Even as you have used gelatin at step 4 so during air drying at step 5 it can increase chances of bacterial contamination which azide cant inhibit. In step 6 if you are using xylene/alcohol for tissue dehydration then you should use xylene based mounting media like DPX. Vectashield is water based mounting media containing glycerol so there's no need to dehydrate your tissue. your choice of mounting media must be in accordance with your last use solvent. Mounted slides should be stored at 4 °C.
1) If you're using PFA, don't do a sucrose saturation.
2) If you're going to do a sucrose saturation, start with a 5% sucrose solution then a 30%.
3) Finally, as Ute and Nabanita have suggested, use SuperFrost + slides, skip the vectashield, and keep slides in the fridge for storage.
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