I am having troubles with the paraffin embedding. First, I am incubating my alginate in 4% PFA overnight, the next day I wash them in PBS then put them in 70% Ethanol. We have an automated machine Histokinette run for paraffin infiltration. When I come to do my paraffin blocks, the alginate hydrogels are always DRY, solid, and way too small than what I had in the beginning. I tried to embed the alginate in Agarose but similarly, the next day, the whole agarose block lost shape and alginate beads cannot be recognized. Should I switch to 2% PFA + sucrose and CaCl2? I am not using that; also, I am using 1% Alginate. Could that be a problem? And does 1% Alginate have an expiry date? I am using the one prepared from last year; but now we have received new Alginate from Novamatrix.
Many thanks,
Patrick
I have no experience with your special problem, but alginate seems to be a very aequoues compound. Paraffin embedding is dehydration pure. So it should be normal to loose part of size.
Therefore fixation should stabilizes the scaffold. I recommend prolonged fixation and reduction of time in high percent ethanols. The longer the dehydration the harder the tissue.
I don't know if it will solve your problem, but you might be more successful using Carnoy's fixative instead of PFA. For certain other hydrated structures, such as mucus, this generally gives far superior results.
It sounds like the solution to your problem is more simple than changing fixatives or percent solutions. I would not recommend changing to 2% PFA because once you get your block working, your specimen will be overexposed. The most important thing is to keep it cool. When you are embedding, you should use a good conduction source to really solidify the paraffin. Just keep everything cold (even on ice if you don't have a cool plate) until you are ready to cut.
- Badges
- Science topic
- Similar topics
- Biological Science
- Zoology
- Invertebrate Zoology
- Arthropods
- Insects