Mouse Sprinal Cord and Brain Tissue

[elenapan]

I'm working on prenatal mice tissue, usually E10-E12.
The tissue is very fragile and doesn't like most of the antigen retrieval methods I've used.
I've tried using 0.1% SDS for 3 minutes before the primary antibody incubation (nuclear) but that seems to completely destroy the tissue.
I've also tried Tris-EDTA pH9 at 95-100C on the slides but I still get the same results. Since we use a 4% PFA fixation (from 40-90minutes depending on stage) followed by glucose and then OCT embedding I don't think there's much I can do to improve the staining. I've also tried EN BLOC antigen retrieval on 11.5 day also embryos but that completely shattered the connective tissue.
The antibodies I'm trying to force to work are supposed to be very specific and were custom made, we have about 5 but they all seem not to be working.
Also, I'm using charged slides to collect the sections on and allow over night drying period.
Any advice?

Thank you for your time,

[excalibur]

First, 40-90 minutes in PFA only fixes the peripheral tissue. The center is still raw. A mouse brain needs several days to completely fix. If you are using frozen sections, antigen retrieval is a needless step. Are you doing a fixation step on the slides after they are cut. Methanol, acetone, PFA, etc. If not you are killing the antigens with the first few steps of your procedure.

Please post your staining protocol.

[elenapan]

This is the actual protocol I'm using which works just fine with almost all our other antibodies

Day 0

1.   Dissect mouse embryos in PBS on ice.
2.   Fix embryos at 4oC in 4% fresh PFA(see note)  (E11.5=1 hr, E10.5 = 45 mins) on gentle shaker.
3.   Rinse with PBS and do another wash with PBS for 1 hr (on ice)
4.   Transfer embryo to 30% sucrose (see note) at +4oC on shaker over night.
5.   Mount in OCT (store at –80oC).


Day 1:
   
1.   Section on superfrost plus slides. Allow to dry for 1 hr (not necessary)
2.   Draw a line with a PAP pen on the slide above the sections.
3.   Cover the sections with 200 μl blocking solution and let stand for 10 minutes or more.
4.   Make the primary antibody dilution in blocking solution meanwhile. Use 200 μl per slide and incubate in a moisted chamber over night at 4ºC.

Day 2:

6.   Wash the slides 3-4 times in 500 μl PBS over a time-span of 20 minutes.
7.   Make the secondary antibody solution (dilute secondary AB in blocking solution). Add at a concentration of 1/2000 (depends which antibody you are   using). Use 200 μl on each slide. Put the slides in the dark and incubate at room temperature for 60-75 minutes (longer has a tendency to give higher background)
8.   Wash 4 times in PBS for 20 – 30 minutes. Try to avoid exposure to light.
9.   Mount the slides: Take all the PBS off from the backside of the slide with a tissue.
10.   Add a little Hoechst on each slide to stain the nucleus. (optional)
11.   Wash slides in PBS to remove excess Hoechst. (optional)
12.   Apply 2-3 drops of mounting media on a cover glass. Then place the slide on top of the cover glass and press a little so the mounting media spreads all around the slide. Try to avoid air bubbles and remove excess liquid by pressing the edges of the slide against a tissue.
13.   After 1 hour apply clear nail varnish on the top and bottom of the slide to fix the cover glass.
14.   After 30 mins the slides are ready to be looked at or the slides can be stored the in darkness at -20 ºC for long term.

[elenapan]

Also, I allow the slides to dry over night at room temperature before using them instead of letting them dry just for a few minutes.

[richard03]

I agree with excalibur that you need longer fixation time.

If you fixed embryo with PFA, why not proceed with paraffin embedding?

If you need or only can do frozen sections, I suggest not to fix with PFA. Instead, fresh frozen the embryos, frozen sectioning and then fix slides with cold acetone.

Hope this helps.

[elenapan]

After thinking about things I've decided to give it a go using paraffin embedding and then proceed with the antigen retrieval step -and hope for the best -
Thank you very much for your help.