20180319 IgG IHC in swine sciatic nerve (Zika Willison+unit patients)

SAMPLES 

We will test the Zika samples from Dr. Willison’s lab and 4 samples from our ICQ database.

ICQ samples:

• 18-612 Guillem Mateo Nieto
• 18-613 Martia Luz Gaspar Rodríguez
• 18-615 San Sebastián López
• 18-617 Miguel Ángel Sorli Peña

PROTOCOL

We will use the sandard protocol:

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

• 003 mielina nodal+/tosco +/linear transversal +
• 004 tosco +/linear transversal +
• 005 tosco++
• 006 mielina axonal ++
• 008 nucleos +++/filamento-fibroblasto +++
• 010 tosco +
• 011 puntiforme +
• 013 nucleos ++ repetir on hold
• 021 Negativo
• 024 puntiforme ++
• 025 Repetir on hold
• 18-612 Repetir on hold
• 18-613 tosco +
• 18-615 mielina axonal ++/linear transversal ++
• 18-617 nodal puntiforme ++
• hvh1 paranodal +++