- Download an up to date .pdf version of the whole-mount-staining-bench-protocol-methanol-dec-2016
- See the log of protocol updates to check which version you have, and why it was eventually changed
- Download files for 3D printed imaging chambers. These can be used for imaging with a 2-photon or confocal microscope.
Recommendations for sample handling
Times and volumes given are a general guideline and could be slightly shorter or longer for specific applications. We recommend trimming the sample to a size most relevant for the specific biological question to insure the best staining and imaging conditions.
We recommend against using multi-well plates for the staining procedure. While convenient, the sample will be in more contact with air, which will degrade the quality of the tissue background. We use 2mL Eppendorf tubes for most incubations (1.6mL of solution+sample), and 5mL Eppendorf tubes for large samples (whole mouse brains) and for washes of all samples.
|Sample type (Mouse)||Incubation time for Ab (n=)||Antibody solution volume||Wash solution volume|
|E18 embryo (whole head)||4d||4ml||4ml|
|Adult hindbrain + cerebellum||3d||2ml||2ml|
|Forebrain (without cortex)||3d||2ml||2ml|
|Whole brain||4 to 6d||4ml||4ml|
To assess the methanol compatibility of untested antibodies, we recommend doing the following:
- Collect 20µm frozen sections of the PFA fixed tissue of interest on superfrost slides.
- Incubate the slides for 3h in 100% methanol
- Rehydrate in PBS directly and proceed with the immunostaining normally. Use non methanol treated slides as a positive control.
If the antibody yields a good signal to noise ratio, the antibody is then compatible with the methanol pretreatment and should work well in whole-mount. If the signal is strongly diminished after the methanol pretreatment, one can use the non-methanol protocol, or test alternative antibodies against the target protein.