Here is a log of changes we have made to the protocol in our quest to produce the best possible results.

December 2016 (Download)

The pre-extraction step has been updated based on the feedback from new users, which sometimes could fail to extract enough lipids in brain tissues prior to the immunostaining. This usually leads to increased background, decreased transparency and decreased antibody diffusion due to excessive oxidation. We believe this is due to users moving too fast through the dehydration steps.

• To make the protocol more foolproof, we added a DCM/MeOH pre-extraction prior to the immunostaining, similar to the one present at the clearing step. In our tests, it didn’t affect the epitopes further, and helps to decrease the background and improves antibody diffusion, specifically in myelinated structures.
• This steps increases the length of the protocol, so if the Mai 2016 already works for you, this new version is optional.

May 2016 (download)

The protocol has been changed to better preserve the morphology of the samples:

• H20 replaces PBS for the dehydration and rehydration steps. This contributes to improve the morphology, as the sample shrinks less during the dehydration.
• THF is removed from the clearing protocol, and replaced by a 2:1 mix of DCM and MeOH (respectively)
• A full list of the reagents we use is provided to help troubleshoot the protocol

January 2015 (download)

 Methanol dehydration updated. We have had issues with rapid volume changes in this step causing tearing of internal structures in adult mouse brain. This has been eliminated with the current dehydration.

·Expanded MeOH gradient and decreased steps to 30min.
·Removed DMSO from steps.
·Removed 20%DMSO/MeOH incubation.

November 2014 (download)

·”Recommendations for sample handling” section reworked.

·Updated notes for simplicity.
·Removed processing time from chart for simplicity. Processing time can be easily calculated from the information given.
·Added time/volume for whole brain.

·Decreased PBS wash in “sample collection to 30min.

·1xPBS/0.2%TritonX-100/20%DMSO/0.3Mglycine was under “immunolabeling” increased to n/2 days where n is incubation time in primary.

·Added reminder on how much secondary to use in “immunolabeling” step 6.

·removed note on agarose embedding from Clearing section for somplicity. We still recommend this for difficult to handle samples.

Original Protocol (download)