SAMPLES (Neuropatías paraneo Clínic)
- D3) 00-1248
- D4) 90-92
- D5) 04-726
- D6) 92-38
- E1) 98-164
- E2) 03-838
- E3) 05-409
- E4) 04-1323
- E5) 16-3512
- E6) 01-203
- F1) 93-94
- F2) 97-506
- F3) 02-616
- IRB 15-467
- AMC 15-107
- prueba anticuerpo comercial NCAM (Mouse CD56/NCAM Ref. 347740 BD) 1/40 con suero ZN018a (lab id nº 13) Zika-GBS por IgM
PROTOCOL
Before the experiment:
– Prepare 1x PBS
– Prepare 250 mL of 5% Goat serum (filtered)
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% blockade 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%.
* LA PRUEBA DEL AC NCAM SE REALIZO CON EL AC COMERCIAL (Mouse CD56/NCAM Ref. 347740 BD) 1/40 en goat al 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- IgM-594 (red) 1/1000 with 5% goat serum in the same microtube.
* LA PRUEBA DEL AC NCAM SE REALIZO CON LOS SECUNDARIOs GAM488 1/1000 + GAH- IgM-594 1/1000
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
- D3) 00-1248: + lin transv
- D4) 90-92: + lin transv
- D5) 04-726: + lin transv
- D6) 92-38: + lin transv
- E1) 98-164: + lin transv
- E2) 03-838: + lin transv
- E3) 05-409: ++ lin transv/++mielina
- E4) 04-1323: + lin transv/+nucleos
- E5) 16-3512: + lin transv
- E6) 01-203: nucleos ++
- F1) 93-94: + lin transv
- F2) 97-506: + lin transv
- F3) 02-616: + lin transv
- IRB 15-467: + lin transv
- AMC 15-107:nucleos +++
- prueba anticuerpo comercial NCAM: parece que el marcaje va contra restos inespecíficos de la preparación. Como en esta prueba no se ha puesto el comercial de NF, aunque se intuye que el nervio está preservado, no podemos confirmar si los resultados se deben a un posible mal estado del nervio. En futuros experimentos, haremos una doble IHC con los comerciales de NF y NCAM. También consultaremos contra qué región va el ac comercial de NCAM (por si hace falta un paso de permeabilización más intenso que el de la acetona) y buscaremos en la bibliografía cómo hacen otros las IHC de NCAM en nervio.
