On various FITC stains (nephrin, CD68 ED-1, Tgfb-1) I run into this issue:
After applying the FITC stain, Evan's Blue and DAPI (in order to observe green, red and blue channels) I place the slides under the microscope immediately after finishing. I will take a few pictures using NIS Elements that will turn out looking perfect. Then when I return to take more pictures the next day, the slides will look as if there is a thick layer of FITC over the tissue on the slide.
Could anyone please tell me what I am doing wrong? I feel like I am washing the slides REALLY well before adding DAPI. Am I not letting them dry long enough before adding the DAPI??
Your help is appreciated!
Below is a picture of the same slide, taken one day apart. You can see how saturated the tissue is and even the surrounding area. The first picture has three merged channels, but you can see that it is very clear and hard to believe it's from the same slide.
Could you provide your staining protocol? Also, I'm unable to see your images. More information is critical to providing insights to the problem.
It seems from your description that you may not be properly mounting the samples. What are you using to mount the samples? It looks like the mountant and/or tissue has degraded and bubbles have formed.
Are you fixing the samples before mounting?
Also you did not mention how you are storing the slides, in the dark at 4C would be best.
Dear Justin: as Jeffrey did I too ask for your staining protocol, especially AFTER staining (you wrote: 'immediately after finishing [staining] I place the slides under the microscope': you didn't use at that point a cover slip, did you? And after that first image taking: what did you do with the object slide carrying the stained sections?):
Idea: Mountant - segregation/decomposition of mounting medium - dehydration (or stained sections drying out) problem...
Edited some minute past original posting: naturally, not to forget, i wanted to mention also Matthews reply (since he might point to the right direction too...(:-))...Matthew, please, apologize for my lapsus...
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining