Hi , I am looking for a full protocol for immunostaining of mouse pancreatic islets after isolation. I put them in RMPI after isolation. How can I prepare them for staining? and can I do costatining with two antibodies?
Yes, you can definitely do double or co-staining with two primary antibodies followed by two appropriate secondary antibodies conjugated to different fluorophores (e.g. Alexa Fluor 488 and Alexa Fluor 568). Just make sure the primary antibodies are from different host species. And a proper control should be included.
Here are some tips for immunostaining mouse pancreatic islets after isolation:
- After isolating the islets, fix them in 4% paraformaldehyde for 30 minutes at room temperature. This will preserve the tissue and allow for antibody staining.
- Wash the fixed islets 2-3 times in PBS to remove excess fixative.
- Permeabilize the islets by incubating in 0.1% Triton X-100 in PBS for 15 minutes. This allows antibodies to penetrate inside cells.
- Block with 5% normal serum (from host species of secondary antibody) for 30-60 min to prevent nonspecific binding.
- Incubate with primary antibody diluted in blocking buffer overnight at 4°C. Good options for islet staining include insulin, glucagon, somatostatin.
- Wash 3 x 5 min in PBS.
- Incubate with fluorophore-conjugated secondary antibody for 1-2 hours at room temperature, protected from light.
- Wash 3 x 5 min in PBS.
- Mount coverslips using fluorescence mounting medium.
Thanks Salah A.Alshehade
I want to understand do I have to do them in paraffin wax blocks or no? Because they will be suspended in media or washing buffer.
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