Immunostaining

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Staining with either antibodies or dyes is performed following sample clearing and a two day PBST buffer wash. Afterwards, sample imaging using three-dimensional techniques, such as confocal microscopy, can be performed. Tissue samples can then be stored in PBST or processed for additional rounds of immunostaining and imaging.

Contents

Guidelines for immunostaining

Successful immunostaining results are often strongly dependent on the antibody choice. Protocol modifications and optimizations may need to be performed for different antibodies, and so one standard procedure can not be applied. The following are guidelines for obtaining successful staining results.

Staining parameters

  • Use long enough incubation times - Multiple days for incubation will likely be needed. For a 1 mm section, a good starting point is: 2 day incubation in primary antibody, 1 day wash with buffer, 2 day incubation in secondary antibody, 1 day wash with buffer.
  • Use high antibody concentrations - Start with antibody concentrations in the range of 10 μg/mL (1:100) to 20 μg/mL (1:50), and lower as needed.
  • Use high temperature - Incubating at 37°C will promote quicker antibody penetration into the clarified tissue sample. If antibody binding is a problem, though, incubation at lower temperatures (4°C, for example) can be done for longer times. A combination of temperatures (37°C followed by 4°C) may combine the benefits of both.
  • PBST buffer - While the sample can be transferred to other buffers for immunostaining, PBST buffer is known to work well.


Other recommendations

  • Passive clearing - Immunostaining seems to work best in samples that have been passively cleared as opposed to ETC. It is recommended to use slightly longer passive clearing incubation times to get the sample to near transparency even in the clearing solution.
  • Smaller sections - 1-2 mm thick sections will provide the best results. Whole tissue staining will need signficantly longer incubation times and antibodies may still not be able to fully penetrate to the core of the sample (a whole mouse brain, for example, took 2 weeks incubation in each antibody with 1 week long buffer wash steps).
  • Blocking agent - Use of a blocking agent in brain tissue has not shown an obvious difference in immunostaining results and is probably not needed. This may not be the case for other tissue types.
  • Hydrogel solution modification - The hydrogel solution can be modified to reduce the amount of crosslinking in the hydrogel-embedded tissue and therefore allow faster and better penetration of antibodies during staining.


Recommended antibodies and dyes

The following is a list of antibodies and dyes that should provide reliable staining results. These recommendations may be useful to start with when testing out the CLARITY method for the first time.

  • Parvalbumin (abcam)
  • Tyrosine Hydroxylase (abcam)
  • Propidium Iodide
  • DAPI (may wash out in mounting solution after 1-2 days)
  • Neurofilament NF-H (chicken antibody from Aves)


Primary antibodies used in CLARITY literature

The following table details primary antibodies that have been used on CLARITY or PACT (passive CLARITY technique) tissue samples in the published literature. It is meant to provide guidance for those unsure of which antibodies may provide good immunostaining results in their CLARITY-processed samples. For more information on how a specific antibody was used and the results obtained, check the corresponding reference (listed in the Journal Articles section).

Antigen Dilution
(vol. antibody soln : vol. buffer)
Tissue type Tissue thickness (mm) Vendor Catalog number References
2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) 1:300 Mouse brain 1 Millipore MAB326 Lee et al, Scientific Reports 2016; Saul et al, Genome Research 2017
Sigma c5922 Lee et al, Scientific Reports 2016
Acetylated tubulin 1:500 Mouse brain; mouse testis; mouse lung 1 Sigma T7451 Lee et al, Scientific Reports 2016
Active caspase-3 1:500 Mouse brain 1 Cell Signaling Technology 9661 Lee et al, Scientific Reports 2016
Alpha-adducin 1:200 Abcam ab51130 Treweek et al, Nature Protocols 2015
Alpha-smooth muscle actin 1:50 Mouse pancreas 1+ Abcam ab5694 Lee et al, BMC Develop. Bio. 2014
1:200-1:300 Mouse mammary gland 1 Abcam ab5694 Lloyd-Lewis et al, Breast Cancer Res. 2016
Alpha-synuclein 1:50 Human brain 3 Becton Dickinson 610787 Liu et al, Neuropathol. Appl. Neurobiol. 2016
Anti-Mullerian hormone (AMH) 1:50 Mouse ovary Santa Cruz Biotechnology sc-6886 Feng et al, Scientific Reports 2017
Aquaporin-4 (AQP4) 1:100 Mouse intestine Santa Cruz Biotechnology sc-20812 Neckel et al, Scientific Reports 2016
Aromatase (CYP19) 1:50 Mouse ovary Abcam ab35604 Feng et al, Scientific Reports 2017
B-cell lymphoma-2 (Bcl-2) 1:250 Mouse brain 1 Santa Cruz Biotechnology sc-7382 Lee et al, Scientific Reports 2016
Beta-amyloid 1:100 Mouse brain 1 Cell Signaling Technology 2454 Murray et al, Cell 2015
Beta-amyloid (A beta) 4G8 Human brain 0.5 Covance Ando et al, Acta Neuropath. 2014
Beta-catenin 1:500 Mouse brain 1 Becton Dickinson 610153 Lee et al, Scientific Reports 2016
Beta-spectrin II 1:200 BD Biosciences 612563 Treweek et al, Nature Protocols 2015
Beta-tubulin 1:200 Santa Cruz Biotechnology sc-9104 Treweek et al, Nature Protocols 2015
Beta-tubulin III (Tuj1) 1:350 Mouse brain 1 Covance MMS-435P Lee et al, Scientific Reports 2016
1:100-1:300 Mouse brain; mouse spinal cord 1 Sigma T8660, T2200 Lee et al, Scientific Reports 2016; Lai et al, PLoS One 2016
1:400 Mouse intestine; human intestine Covance PRB-435P Neckel et al, Scientific Reports 2016
1:1000 Organoid Promega G712A Lee et al, Neuropsychopharmacology 2017
1:50 Mouse ovary Abcam ab18207 Feng et al, Scientific Reports 2017
1:100 Mouse pancreas; human pancreas 1+ Millipore MAB1637 Hsueh et al, Scientific Reports 2017
BH interacting domain death agonist (BID) 1:25 Mouse ovary Santa Cruz Biotechnology sc-292494 Feng et al, Scientific Reports 2017
Brain-derived neurotropic factor (BDNF) 1:50 Mouse ovary Abcam ab72439 Feng et al, Scientific Reports 2017
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Multi-round molecular phenotyping of hippocampus slice

Multi-round phenotyping

The stable framework of the clarified tissue, whereby the innate biomolecules are chemically attached to the hydrogel network, enables multiple rounds of molecular phenotyping without damage to the preserved structure. In fact, the clearing solution containing SDS micelles that was originally used for tissue lipid clearing can be used following the first round of immunostaining to denature antibodies, disrupt binding, and wash the first round of antibodies and molecular labels out of the hydrogel-embedded tissue.

Procedure

  • Remove sample from imaging chamber, place in 50 mL PBST and incubate at room temperature or 37°C overnight with shaking
  • Place sample in 50 mL of clearing solution and incubate at 60°C overnight with shaking to wash out first round of antibodies
  • Place sample in 50 mL PBST and incubate at room temperature or 37°C overnight with shaking to wash out clearing solution
  • Complete next round of immunostaining and imaging