To test whether 4% PFA -fixed (10 min) and 4% PFA-fixed (10 min)+ 5% Goat-blocked (1 hour) teased swine sciatic frozen nerves perform better in IHC experiments when compared to straight-frozen teased nerves.
SAMPLE
Positive ct HVH1 (CNTN1+)
PROTOCOL
We will use the sandard protocol, however:
- In 4% PFA-fixed teased nerves: no acetone fixation will be performed (skip steps 2-6)
- In 4% PFA-fixed + 5% Goat-blocked teased nerves: no acetone fixation (skip steps 2-6) or extra blockade will be performed (skip step 10)
1) Prepare a humid chamber with dampened sponges in an opaque box.
2) Prepare a Hellendhal staining bucket full of acetone (-20 ° C).
3) Remove the swine sciatic nerve teasing of the freezer and quickly put it directly into acetone. Note: Do not remove the box from the freezer. Proceed to sample fixation for 10 min at RT. (If summer locate the Hellendhal chamber in a large bucket with ice).
4) Recycle acetone and cover the bucket with 1x PBS. Pour PBS at the corners, not directly over the sample.
5) Wash approximately 5 min with agitation (about 35 rev / min).
6) Repeat the washing step.
7) Dry the sample with a tissue without touching the actual sample.
8) Use the marker to highlight the location of the sample without exerting excessive pressure. Note: it is important to quickly cover the marker just after using it.
9) Indicate the sample number and the date in pencil on the frosted glass and place the samples in the humid chamber.
10) With a plastic Pasteur pipette cover the entire sample with Goat Serum 5% blocking solution . Incubate 1 hour at RT in a humid chamber .
11) Dilute the serum samples 1/100 in 5% goat.
12) Wipe the sample dry with tissues and gently remove excess blocking solution with Whatman paper.
13) Add 200 mcL /sample of diluted serum and incubate 1 h at TA in the humid chamber.
14) Wash approximately 5 min with agitation (about 35 rev / min).
15) Repeat the washing step.
16) Dilute commercial primary antibody Chicken anti-human / rat / mouse-NF (AF3235 R & D System) * 1/500 with goat serum 5%.
17) Wipe the sample dry with tissues and gently remove excess blocking solution with Whatman paper.
18) Add 200 mcL /sample of the commercial diluted antibody and incubate 1 h at TA in the humid chamber.
19) Wash approximately 5 min with agitation (about 35 rev / min).
20) Repeat washing step twice.
21) Prepare the mixture of secondary antibodies. Dilute GAC488 antibody (green) 1/2000 and GAH- IgG-594 (red) 1/1000 with 5% goat serum in the same microtube.
22) Add 500 mcL/ sample of the mixture of secondary antibodies and incubate 1 h in a humid chamber at TA.
23) Wash approximately 5 min with agitation (about 35 rev / min). Important: avoid contact with light.
24) Repeat the washing twice, covering the sample.
25) Wipe the sample tissues and eliminate most of the excess blocking solution with Whatman paper.
26) Mount the preparation with Fluoprep: add two Fluoprep drops per sample and sealed them with a coverslip. Press to remove the excess and to ensure the homogeneity of the preparation. Carefully clean the edges and store protected from light. Note: before adding the Fluoprep drops to the sample, pour a couple on a piece of paper to remove bubbles.
RESULTS
The standard procedure enables a better analysis of the teased nerve. Nerve integrity seems to be compromised in the other two conditions, most likely due to sample degradation that may be caused by freezing samples after being submerged in liquid (100% sample drying is hard to achieve in such conditions, and this followed by a freezing step might be accountable for the lack of conservation seen in these samples).
In following experiments we will try to further air dry the fixed and the fixed+blocked samples and we will additionalyy test whether acetone might be a better fixating agent than PFA.
