E10.5 embryos were fixed in 4% PFA in PBS at 4°C, rinsed in PBS, then permeabilized in PBS with 0.5% Triton X-100 for 24hrs at 4°C. Neurofilament immunodetection was performed as previously described (Ray et al., 2020 (link)). Primary anti-neurofilament antibody (DHSB, 2H3) was used at a 1:20 dilution and anti-mouse HRP-conjugated secondary antibody (Jackson ImmunoResearch, 115-035-003) was used at 1:1000 dilution. Signal was developed using the ImmPACT DAB Substrate Kit (Vector Laboratories, SK-4105). Photographs were taken using a Nikon SMZ-U dissecting scope fitted with a Jenoptik ProgRes C5 camera.
Protocol detail
Neurofilament Immunodetection in E10.5 Embryos
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anti-mouse HRP-conjugated secondary antibody
ImmPACT DAB Substrate Kit
SMZ-U dissecting scope
ProgRes C5 camera
Anti antibody
Dilution
Embryos
Mouse
Neurofilament
Triton x 100
Vector
Corresponding Organization : Icahn School of Medicine at Mount Sinai
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Variable analysis
independent variables
- Tissue fixation in 4% PFA in PBS at 4°C
- Permeabilization in PBS with 0.5% Triton X-100 for 24hrs at 4°C
- Neurofilament immunodetection
- E10.5 embryos
- Rinsing in PBS
- Primary anti-neurofilament antibody (DHSB, 2H3) used at 1:20 dilution
- Anti-mouse HRP-conjugated secondary antibody (Jackson ImmunoResearch, 115-035-003) used at 1:1000 dilution
- Signal development using ImmPACT DAB Substrate Kit (Vector Laboratories, SK-4105)
- Photographs taken using a Nikon SMZ-U dissecting scope fitted with a Jenoptik ProgRes C5 camera
- Not specified
- Not specified
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