Been struggling with a slide mounted IF protocol. Here are my parameters:
1) Fixed in 4% PFA for 2 hours at 4 degrees C
2) 10um cryosections air dried on slides O/N
3) Coplin jar water bath to remove gelatin
4) Draw circles around sample with pap pen
5) Blocking buffer composed of 2% BSA and 10% DS in PBS w/ Triton x100 (PBST)
6) Primary at 4 degrees O/N
7) Wash 3x in PBST
8) Secondary for 2 hours at RT
9) Wash 3x in PBST
10) Mount with brand new Vectashield w/DAPI stain
When I go to image the sections most of samples have no DAPI signal. The few that are stained have the DAPI signal localized to the center of the section. Furthermore, this tissue has endogenous GFP reporter that is almost completely quenched. Any ideas for what might be happening here? I can send pictures if it would help.
Hi,
You may try to add DAPI on the secondary AB step. Also you may try to use Mowiol 4-88 for mounting. Alternatively, you may try to use Hoechst instead of DAPI.
Best,
Michail
Dear Colton,
you might did something wrong at the last step while you are covering the slides with coverslip make sure you are not moving away DAPI drops. also you could use DAPI with 1/1000 concentration for 15 min before going for vectshield step it would give a very bright and shiny DAPI image. also One hour is enough for secondary AB.
If you are using confocal try to arrange the laser before starting with the main slides. High power of laser will destroy the tissue sections and photo bleaching will happen.
Good Luck
Masoumeh
What kind of tissue is it? Sounds like bad fixation to me.
Are you cutting the tissue without fixation?
If so, how? Vibratome? cryo?
All these information will be nescessary o offer you help.t
two obvious problems here: gfp is easily quenched by PFA unless it is a "hardened" variant (e.g., ZsGreen) so you may have to immunostain for GFP. Vectashield DAPI was originally designed only for cell biology so you may have not enough DAPI to stain your nuclei if you have large sections and/or multiple sections per slide, so, as michail suggested, add dapi to the II Ab step (or change to far-red dna stains: far better!). the bright center spot: this is unclear till we see it...
I would do an H&E or Cresylviolett stain (brain tissue) to check weather the tissue looks fine. I suppose, like Arghya, bad fixation. 2 possibilities to improve the qualitiy: 1. use unfixed cryo sections and fix them after drying in 4% PFA . 2. fix your tissue block for several days in 4%PFA (1 qcm ca 3-4 days) cryoprotect the tissue in 30% sucrose until the block sanks on the bottem of the vial (same size same duration). Which one should be the best depends on stainings and antibodies you like to use afterwards.
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