I'm currently working on the FFPE slice culture model and have some difficulties in making sure that the antibodies introduced to these samples are bound correctly on them. Besides that, I also face a problem in maintaining the section on the slides as some of the samples are absent especially after the antigen retrieval step.
For your information, I'm using gelatin-coated slides for this IF.
Any suggestion is really meaningful. Thanks in advance for your help.
We are using Starfrost Advanced Adhesive Slides for all of our immunofluorescence and I never had FFPE sections (3 microns) detach, even during 20 min citrate buffer boiling for antigen retrieval. How thick do you cut your samples?
Regarding the cutting procedure: the precooled FFPE blocks are cut at a microtome, put into a warm (45 - 65 °C depending on the tissue) waterbath and from there gathered onto the glass slide. Then dried over night at 37 °C in an incubator (also 2 days at room temperature possible, or - supposedly - 1 h at 70 °C, but I never tried that). Then either store them (at least up to 1 month should be fine), or stain right away.
Regarding the binding of your antibodies: does the supplier say that your antibody works on FFPE sections of your species of interest/ is there published literature with your exact combination? If yes, you could try to change the pH of your retrieval buffer, or retitrate the antibody. Otherwise: order another antibody^^
In case I misunderstood any of your questions, feel free to provide more details :-)
What is the thickness of slice? It must be thicker and make sure to keep it wet all the times
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology
- Sectioning