immunofluorescence

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namratta
namratta's picture
immunofluorescence

I have to do immunofluoroscence of pERK and p-p90RSK from CST  on freezing microtome cut sections;paraformaldehyde fixed ;from mice brain(28-30 g mice)

  1. I plan to go for 4% PFA in PBS
  2. Post fixation 2hrs in same fixative
  3. Cryoprotect in 30% sucrose from 6-12 hrs(let me know the time for adult mice)
  4. 30% sucrose made in PBS; should i add ehthylene glycol for cutting on freezing microtome the brain slices
  5. How i store brains sections for a month
  6. should i go for floating method or on glass slides; which thickness would the best FOR THESE 2 PHOSPHO PROTEINS. any special suggestion for staining phosphoproteins
  7. Could plz correct if i am wrong in any way or plz give yr kind experienced suggestions

thanks

Arvind Singh Pundir
Arvind Singh Pundir's picture
Hi Namratta,here are answeres

Hi Namratta,
here are answeres to your queries

  1. yes 4% PFA is ok
  2. post fixation for 2 to 4 hrs is  also ok
  3. in 30% sucrose at 4 degree celsius, you should keep the tissue till it sunks to the bottom of  container
  4. you can go in increasing conc of sucrose like 20% and then 30% for more efficient cryoprotection
  5. if you want to store sections for long then use anti freeze solution(30% glycerol or 30% ethylene glycol in PBS) with sod azide and store at -80 or -30 celsius
  6. floating sections are better for IHC if possible, as there is more surface area exposed to the reaction mixture
  7. take 20 to 30 micron sections, rest is fine you can share your protocol for getting any tips

Good luck and cheers for SSI