I have GD18.5 heart tissue currently snap frozen in -80, apart from mRNA and protein analysis on whole tissue, is there a way of isolating single cell populations to study localization of a protein / transcript?
You will not be able to isolate individual cells from frozen tissue as they will lyse upon freeze/thawing.
One method could be doing laser capture microdissection using paraffin-embedded formalin fixed tissue sections, or specially prepared frozen tissue sections to isolate the cells of interest for proteomic analysis by mass spectrometry. There are a lot of papers in the literature describing these methods, but you do need a special laser-equipped microscope and facilities to prepare the tissue blocks and do the sectioning.
Hello,
You can isolate cardiac fibroblasts since these cells are quite sturdy and can withstand the freeze/thawing.
However, when it comes to cardiomyocytes, you have two scenarios: if you are dealing with neonate tissues, you may have a chance to isolate the cells by "stir cell", but for adult tissues it is preferable to mount the freshly isolated heart on a Langendorff apparatus in order to isolate highly pure cardiomyocytes.
For further info, you can check these papers:
- For fibroblasts
https://pubmed.ncbi.nlm.nih.gov/30318928/
- For adult cardiomyocytes
https://pubmed.ncbi.nlm.nih.gov/25213556/
Good luck
Hi Cara,
Ideally you wouldn't isolate cells from frozen tissue but rather presented with a frozen heart you'd follow an embedding and sectioning method as described by Mary Cristine Charlesworth above.
However, if the isolation of single cells is absolutely necessary then it is possible to isolate myocytes from frozen tissue, though with a lower yield than fresh tissue. See both Article Cardiomyocyte proliferation contributes to heart growth in y...
andArticle Methods for isolation and transcriptional profiling of indiv...
for how to do so.Good luck
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