Oct 26, 2020
3D Immunostaining for CLARITY-processed samples
- Seth Currlin1
- 1University of Florida
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: [email protected]
Protocol Citation: Seth Currlin 2020. 3D Immunostaining for CLARITY-processed samples. protocols.io https://dx.doi.org/10.17504/protocols.io.bnzdmf26
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 26, 2020
Last Modified: October 26, 2020
Protocol Integer ID: 43781
Keywords: 3d immunostaining for clarity, 3d immunostaining, immunostaining large sample, immunostaining clarity, human thymus tissue, piece of human thymus tissue, large tissue volume, particular tissue, dense tissue type, processed sample, mm3 in size, large sample
Abstract
This is a guide for immunostaining CLARITY-processed samples. dx.doi.org/10.17504/protocols.io.8jihuke
These steps are meant to be a guide for immunostaining large samples and should be optimized to suit your particular tissues and reagents. Large tissue volumes and dense tissue types will require longer incubation and wash times. The parameters suggested below are for a piece of human thymus tissue approximately 5 mm3 in size.
A useful link: http://wiki.claritytechniques.org/index.php/Immunostaining
Materials
Solutions:
Wash buffer (PBST): 1x phosphate buffered saline (PBS), 0.5% Triton X-100, pH 7.4; at least 20x volume of tissue.
Blocking buffer: 1x PBS, 1% horse serum, 0.5% Triton-X 100, 0.05% Tween-20, pH 7.4; at least 10x volume of tissue.
oAnother serum type should be used based on the secondary antibody.
Staining buffer: 1x PBS, 0.5% Triton X-100, 0.05% Tween-20, pH 7.4; at least 10x volume of tissue.
Troubleshooting
Before start
Ensure the tissue is sufficiently transparent for your imaging requirements. If the sample has not been successfully cleared then subsequent imaging will be difficult due to regions retaining light-obscuring lipids. Samples should be imaged prior to immunostaining to confirm adequate clearing, particularly among excitation/emission settings intended for later use.
Immunohistochemistry for large (5 mm3) tissue volumes
3w 5d
Wash out residual clearing solution
Three washes in PBST, 24 hours each, 37C with gentle agitation.
3d
Nonspecific Epitope Blocking
Blocking buffer incubation: three days, 37C with gentle agitation.
Note: Serum type should be used based on the secondary antibody host species.
3d
Primary Antibody Incubation
1:100 dilution (~10 ug/mL) in Staining Buffer.
Primary antibody incubation: five days, 37C with gentle agitation; then two days, 4C with gentle agitation.
1w
Wash out Primary Antibody
Three washes, 24 hours each, at 37C with gentle agitation.
3d
Secondary Antibody Incubation
1:200 dilution (~5 ug/mL) in Staining Buffer.
Secondary antibody incubation: five days at 37C with gentle agitation; two days at 4C with gentle agitation.
Note: Protect samples from light.
1w
Wash out Secondary Antibody
Three washes, 24 hours each, at 37C with gentle agitation.
Note: Protect samples from light.
3d
Prepare for 3D Imaging or Storage
Prepare for imaging or store samples in washing buffer (PBST) at 4C.
For 3D imaging guide, see: https://dx.doi.org/10.17504/protocols.io.begajbse