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
c-Fos 1:500 Mouse brain 1 Santa Cruz Biotechnology sc-253 Lee et al, Scientific Reports 2016
C-Kit 1:200 Mouse intestine Dako A4502 Neckel et al, Scientific Reports 2016
Calbindin (CB) 1:500 Mouse brain 0.5 Swant CB-38 Unal et al, J. Neurosci. 2015
1:100 Mouse brain 1 Cell Signaling Technology 13176 Murray et al, Cell 2015
Abcam ab11426 Murray et al, Cell 2015
Calbindin D-28k 1:100-1:500 Mouse brain 0.25-1 Swant 300 Lee et al, Scientific Reports 2016; Phillips et al, Scientific Reports 2016
Calcitonin gene-related peptide (CGRP) 1:300 Mouse brain 1 Abcam ab36001 Lee et al, Scientific Reports 2016
Calretinin 1:100 Mouse brain 0.5 Swant 7699/3H Unal et al, J. Neurosci. 2015
1:500 Mouse brain 1 Swant 7697 Lee et al, Scientific Reports 2016
1:800 Mouse brain 1 Millipore AB5054 Lee et al, Scientific Reports 2016
Caveolin 1:300 Mouse brain 1 Abcam ab18199 Lee et al, Scientific Reports 2016
CD 31 1:50-1:100 Mouse intestine; mouse pancreas; human pancreas 1+ Abcam ab28364 Neckel et al, Scientific Reports 2016; Hsueh et al, Scientific Reports 2017
1:100 Mouse spinal cord; mouse embryo; rat brain 4 Santa Cruz Biotechnology Woo et al, Exp. Mol. Med. 2016
1:500 Mouse brain 1 Becton Dickinson 557355 Lee et al, Scientific Reports 2016
CD133 1:500 Mouse brain 1 Millipore MAB4310 Lee et al, Scientific Reports 2016
CD34 1:100 Mouse spleen Santa Cruz Biotechnology sc-9095 Epp et al, eNeuro 2015
CD8-PE 1:100 Mouse colon; mouse reproductive tract BioLegend Kieffer et al, eLife 2017
Choline acetyltransferase (ChAT) 1:50-1:200 Mouse brain 0.5-1 Millipore AB144P Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014; Unal et al, J. Neurosci. 2015; Lee et al, Scientific Reports 2016
1:100 Mouse intestine Chemicon International AB144P Neckel et al, Scientific Reports 2016
Collagen type III 1:300 Mouse brain 1 Abcam ab7778 Lee et al, Scientific Reports 2016
Collagen type IV 1:300 Mouse brain; mouse kidney; human spinal cord 1 Abcam ab6586 Lee et al, Scientific Reports 2016
1:100 Mouse lung Millipore AB756P Epp et al, eNeuro 2015
Mouse pancreas; human pancreas 1+ Millipore MAB8201 Hsueh et al, Scientific Reports 2017
Collagen type IX 1:20 Chicken embryo foot DSHB 2C2 Botelho et al, J. Exp. Zool. 2017
Complex IV subunit I (COX1) 1:100 Mouse brain; human brain 0.25 Abcam ab14705 Phillips et al, Scientific Reports 2016
Complex IV subunit IV (COX4) 1:100 Mouse brain; human brain 0.25 Abcam ab14744 Phillips et al, Scientific Reports 2016
Ctip2 1:800 Mouse brain 1 Abcam ab18465 Lee et al, Scientific Reports 2016
Cut-like homeobox-1 (Cux1) 1:150 Mouse brain 1 Santa Cruz Biotechnology sc-13024 Lee et al, Scientific Reports 2016
Cytokeratin 1:400 Mouse intestine Dako Z0622 Neckel et al, Scientific Reports 2016
Cytokeratin-19 (CK19) 1:100 Mouse liver 1+ Developmental Studies Hybridoma Bank Troma-III Font-Burgada et al, Cell 2015
Cytokeratin-20 (CK20) 1:100 Mouse pancreas; human pancreas 1+ Dako Ks20.8 Hsueh et al, Scientific Reports 2017
Cytokeratin-7 (CK7) 1:100 Mouse pancreas; human pancreas 1+ Dako OV-TL 12/30 Hsueh et al, Scientific Reports 2017
Cytokeratin-8 1:50-1:200 Mouse mammary gland 1 Developmental Studies Hybridoma Bank Troma-I Lloyd-Lewis et al, Breast Cancer Res. 2016
Digoxigenin (DIG) 1:500 Mouse brain 1 Sylwestrak et al, Cell 2016
Dopamine transporter (DAT) 1:500 Mouse brain 0.3 Millipore MAB369 Stout et al, ACS Chem Neuro 2016
Dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) 1:1000 Mouse brain 1 Cell Signaling Technology 19A3 Poguzhelskaya et al, Mol. Neurodeg. 2014
Doublecortin (DCX) 1:500 Mouse brain 1 Santa Cruz Biotechnology sc-8066 Lee et al, Scientific Reports 2016
DsRed 1:300-1:500 Mouse brain; Xenopus brain; Zebrafish 3 Clontech Laboratories 632496 Plummer et al, Development 2015; Fini et al, Scientific Reports 2017; Passoni et al, Disease Models and Mech. 2017
Dystrophin 1:100 Mouse skeletal muscle Abcam ab15277 Verma et al, Skeletal Muscle Reg. in the Mouse 2016
E-Cadherin 1:50-1:200 Mouse mammary gland; mouse embryonic epididymis; mouse embryonic kidney; mouse embryonic lung 1 Cell Signaling Technology 3195 Lloyd-Lewis et al, Breast Cancer Res. 2016; Hirashima and Adachi, PLoS One 2015
1:50-1:300 Mouse mammary gland; Zebrafish; rat kidney 1-1.5 Becton Dickinson 610182 Lloyd-Lewis et al, Breast Cancer Res. 2016; Cronan et al, Immunity 2016; Unnersjo-Jess et al, Kidney International 2016
Epidermal growth factor receptor (EGFR) 1:500 Mouse brain 1 Abcam ab2430 Lee et al, Scientific Reports 2016
ER81 1:100 Mouse brain 1 Covance PRB-362C Murray et al, Cell 2015
Estrogen related receptor alpha (ERR-alpha) Mouse brain 0.2 Santa Cruz Biotechnology sc-66882 Saul et al, Genome Research 2017
Excitatory amino acid transporter-2 (EAAT2) 1:100 Mouse brain 1 Abcam ab178401 Murray et al, Cell 2015
Forkhead box protein P2 (FOXP2) 1:100 Mouse brain 1 Abcam ab1307 Murray et al, Cell 2015
Gamma-aminobutyric acid (GABA) 1:500 Mouse brain 1 Sigma A2052 Lee et al, Scientific Reports 2016
Glial fibrillary acidic protein (GFAP) 1:50-1:200 Mouse brain; human brain 0.5-2 Abcam ab53554, ab4674 Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014; Murray et al, Cell 2015; Costantini et al, Scientific Reports 2015
1:400 Mouse brain 1 Aves GFAP Yang et al, Cell 2014
1:100-1:500 Mouse brain 1 Cell Signaling Technology 3670 Murray et al, Cell 2015; Lee et al, Scientific Reports 2016
1:800 Mouse brain 1 Abcam ab7260 Lee et al, Scientific Reports 2016
1:500-1:2000 Mouse brain 0.25-1 Dako Z0334 Lee et al, Scientific Reports 2016; Perez et al, J. Clinical Investigation 2017
1:500 Mouse brain 1 Invitrogen 130330 Lee et al, Scientific Reports 2016
1:50-1:100 Mouse brain 1-2 Sigma Bastrup and Larsen, J. Neuro. Methods 2017
Glucagon 1:100 Mouse pancreas; human pancreas 1+ Takara M182 Hsueh et al, Scientific Reports 2017
Glucagon-like peptide 1 (GLP-1) 1:1500 Rat brain 2 Peninsula Laboratories T-4363 Zheng and Rinaman, Brain Struct. Funct. 2016
Glucose transporter-1 (GLUT1) 1:800 Mouse brain 1 Abcam ab15309 Lee et al, Scientific Reports 2016
1:100-1:300 Mouse brain; human brain 0.25-1 Thermo Fisher PA1-21041 Lee et al, Scientific Reports 2016; Phillips et al, Scientific Reports 2016
Glutamic acid decarboxylase-67 (GAD67) 1:100 Mouse brain 1 Abcam ab26116 Murray et al, Cell 2015
1:500 Mouse brain 1 Millipore MAB5406 Lee et al, Scientific Reports 2016
Green fluorescent protein (GFP) 1:50-1:100 Mouse brain; mouse pancreas; human pancreas 0.3-1+ Life Technologies A21311, A31851, A21312, A31852, G10362, A11122 Chung et al, Nature 2013; Tomer et al, Nature Protocolos 2014; Adhikari et al, Nature 2015; Murray et al, Cell 2015; Li et al, Scientific Reports 2015; Uribe-Marino et al, Biological Psychiatry 2016; Hsueh et al, Scientific Reports 2017
1:200-1:600 Mouse brain; Xenopus brain; Zebrafish 0.5+ Aves GFP-1020, GFP-1010 Unal et al, J. Neurosci. 2015; Fini et al, Scientific Reports 2017; Mayrohofer et al, Disease Models and Mech. 2017; Xavier et al, J. Comparative Neurology 2017; Passoni et al, Disease Models and Mech. 2017
1:100 Mouse lung 0.3 Novus Saboor et al, Eur. Respir. J. 2016
1:500-1:1000 Mouse brain 1-3 Abcam ab13970 Plummer et al, Development 2015; Lee et al, Scientific Reports 2016
1:300 Mouse brain 1 Abcam ab290 Lee et al, Scientific Reports 2016
1:200 Mouse brain 1 Millipore AB3080 Yu et al, Scientific Reports 2017
1:100 Mouse spleen Rockland Immunochemicals 600-101-215 Murray et al, PLoS One 2017
Growth associated protein-43 (GAP43) 1:500 Mouse brain 1 Abcam ab16053 Lee et al, Scientific Reports 2016
Heme oxygenase-1 (HO-1) 1:250 Mouse brain 1 Abcam ab13248 Lee et al, Scientific Reports 2016
Histone H3 1:100 Mouse brain 1 Cell Signaling Technology 4499, 12230 Murray et al, Cell 2015
Abcam ab1791 Murray et al, Cell 2015; Kim et al, PNAS 2015
HIV-1 p24 1:200 Mouse colon; mouse reproductive tract Dako Kieffer et al, eLife 2017
HuC/D 1:50 Mouse intestine Molecular Probes A-21271 Neckel et al, Scientific Reports 2016
Human CD3 1:200 Mouse colon; mouse reproductive tract Dako Kieffer et al, eLife 2017
Human CD4 1:50 Mouse colon; mouse reproductive tract Invitrogen Kieffer et al, eLife 2017
Inositol 1,4,5-triphosphate 3-kinase A (IP3KA) 1:500 Mouse brain 1 Santa Cruz Biotechnology sc-11206 Lee et al, Scientific Reports 2016
Insulin 1:100-1:500 Human pancreas; mouse pancreas 2 Dako A0564 Treweek et al, Nature Protocols 2015; Hsueh et al, Scientific Reports 2017
Integrin b4 1:200 Mouse kidney; mouse heart; mouse lung 1 Santa Cruz Biotechnology sc-9090 Yang et al, Cell 2014
Integrin b5 1:200 Mouse kidney; mouse heart; mouse lung 1 Santa Cruz Biotechnology sc-14010 Yang et al, Cell 2014
Ionized calcium-binding adaptor molecule-1 (Iba1) 1:200 Mouse brain 1 Biocare Medical CP 290A Yang et al, Cell 2014
1:50-1:500 Mouse brain; human brain 1 Wako 019-19741 Murray et al, Cell 2015; Liu et al, Neuropath. Appl. Neurobiol. 2016; Lee et al, Scientific Reports 2016
Keratin-5 1:100 Mouse mammary gland 1 BioLegend 905501 Lloyd-Lewis et al, Breast Cancer Res. 2016
Ki67 1:100 Mouse brain 1 Abcam ab16667 Murray et al, Cell 2015
Laminin 1:100-1:500 Mouse brain; mouse liver; mouse skeletal muscle 1 Sigma L9393 Lee et al, Scientific Reports 2016; Verma et al, Skeletal Muscle Regen. in the Mouse 2016
1:100 Mouse skeletal muscle Sigma L0663 Verma et al, Skeletal Muscle Regen. in the Mouse 2016
Mouse pancreas; human pancreas 1+ Millipore ab2034 Hsueh et al, Scientific Reports 2017
Lectin 1:50 Mouse skeletal muscle Vector Labs DL-1174, DL-1177 Verma et al, Skeletal Muscle Regen. in the Mouse 2016
mCherry 1:150 Mouse nasal septum Acris Schnittke et al, PNAS 2015
MelanA 1:100 Mouse pancreas; human pancreas 1+ Dako A103 Hsueh et al, Scientific Reports 2017
Microtubule-associated protein-2 (MAP2) 1:50 Mouse brain 1 Abcam ab32454 Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014
1:100 Mouse brain 1 Cell Signaling Technology 8707 Murray et al, Cell 2015
Covance PCK-554P Murray et al, Cell 2015
1:100-1:500 Mouse brain; mouse spinal cord 1 Millipore MAB3418, AB5622 Lee et al, Scientific Reports 2016; Lai et al, PLoS One 2016
1:350 Mouse brain 1 Cell Signaling Technology 4542 Lee et al, Scientific Reports 2016
Microtubule-associated protein-2B (MAP2B) 1:300 Mouse brain 1 Becton Dickinson 610460 Lee et al, Scientific Reports 2016
Myelin basic protein (MBP) 1:50-1:300 Mouse brain; human brain 0.5-1 Aves MBP Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014; Murray et al, Cell 2015; Lee et al, Scientific Reports 2016
1:100 Mouse brain 1 Abcam ab7349, ab24567, ab134018 Murray et al, Cell 2015
1:500 Mouse brain 1 Abcam ab40390 Lee et al, Scientific Reports 2016
1:50 Mouse brain 6 Millipore Chang et al, NeuroImage 2017
1:500 Mouse brain; human brain 0.25 BioLegend 836502 Phillips et al, Scientific Reports 2016
1:200 Mouse brain 0.2 Santa Cruz Biotechnology sc-13914 Treweek et al, Nature Protocols 2015
N-terminal EF-hand calcium-binding protein-1 (NECAB1) 1:100 Mouse spinal cord 1+ Atlas Antibodies AB HPA023629 Zhang et al, PNAS 2014
NDUFA13 1:100 Mouse brain; human brain 0.25 Abcam ab110240 Phillips et al, Scientific Reports 2016
NDUFB8 1:100 Human brain 0.25 Abcam ab110242 Phillips et al, Scientific Reports 2016
Nephrin 1:100 Rat kidney 1-1.5 Santa Cruz Biotechnology sc-19000 Unnersjo-Jess et al, Kidney International 2016
Nestin 1:500 Mouse brain 1 Millipore MAB353 Lee et al, Scientific Reports 2016
Neural cell adhesion molecule (NCAM) 1:250 Mouse brain 1 Millipore AB5032 Lee et al, Scientific Reports 2016
Neurofilament 1:100 Human brain 0.5 Dako M0762 Ando et al, Acta Neuropath. 2014; Lai et al, PLoS One 2016
Neurofilament 160 kDa 1:100 Mouse brain; human brain 0.5-1 Aves NFM Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014
Mouse brain 1 Abcam ab92539, ab7794, ab64300 Murray et al, Cell 2015
Neurofilament 200 kDa 1:100 Mouse brain; human brain 0.5-1 Aves NFH Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014
Mouse brain 1 Abcam ab4680, ab8135 Murray et al, Cell 2015
Neurofilament 2F11 1:50-1:100 Mouse brain; rat brain; human brain 3 Agilent M0762 Liu et al, Neuropathol. Appl. Neurobiol. 2016
Neurofilament 68 kDa 1:100 Mouse brain; human brain 0.5-1 Aves NFL Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014
Neurofilament H 1:100-1:300 Mouse brain 1 Cell Signaling Technology 2836BF Murray et al, Cell 2015; Lee et al, Scientific Reports 2016
Neurofilament H 200 kDa 1:200 Mouse brain; human brain 0.25 Millipore AB5539 Phillips et al, Scientific Reports 2016
Neurofilament H SMI-32 1:500 Mouse brain; human spinal cord 1 Covance SMI-32R Lee et al, Scientific Reports 2016
Neurofilament L 1:100 Mouse brain 1 Cell Signaling Technology 2837 Murray et al, Cell 2015
Neurofilament M (NFM) 1:500 Mouse brain 1 Santa Cruz Biotechnology sc-51683 Lee et al, Scientific Reports 2016
Neurofilament SMI-31 1:1000 Mouse brain 0.25 BioLegend 801601 Phillips et al, Scientific Reports 2016
Neurofilament SMI-312P 1:100 Mouse brain 1 Covance SMI-312P Murray et al, Cell 2015
Neurofilament SMI-312R 1:100 Mouse brain 1 Covance SMI-312R Murray et al, Cell 2015
Neuron-glial antigen-2 (NG2) 1:500 Mouse brain 1 Millipore AB5320 Lee et al, Scientific Reports 2016
Neuronal class III beta-tubulin 1:100 Mouse brain 1 Covance PRB-435P Murray et al, Cell 2015
Neuronal nuclei (NeuN) 1:100 Mouse brain 1 Abcam ab104225, ab177487 Murray et al, Cell 2015
Cell Signaling Technology 12943 Murray et al, Cell 2015
Covance SIG-39860 Murray et al, Cell 2015
1:500 Mouse brain 1 Millipore MAB377 Lee et al, Scientific Reports 2016
1:50-1:100 Mouse brain 1-2 Sigma Bastrup and Larsen, J. Neuro. Methods 2017
Neuronal PAS domain protein 4 (NPAS4) 1:3000 Mouse brain 2 Ye et al, Cell 2016
Neuropeptide Y (NPY) 1:500 Rat brain 2+ Millipore AB1583 Zheng and Rinaman, Brain Struct. Funct. 2016
1:100 Mouse brain 1 Cell Signaling Technology 11976 Murray et al, Cell 2015
Nitric oxide synthase-1 (NOS1) 1:250 Mouse brain 1 Santa Cruz Biotechnology sc-648 Lee et al, Scientific Reports 2016
1:400 Mouse intestine Research & Diagnostic Antibodies AS-1657 Neckel et al, Scientific Reports 2016
Olfactory marker protein (OMP) 1:250 Mouse brain 1 Thermo Fisher OSP00001W Lee et al, Scientific Reports 2016
Oligodendrocyte lineage transcription factor 2 (Olig2) 1:300 Mouse brain 1 Millipore AB9610 Lee et al, Scientific Reports 2016
1:250 Mouse brain 1 IBL JP-18953 Lee et al, Scientific Reports 2016
Oligodendrocyte marker O4 (O4) 1:250 Mouse brain 1 Millipore MAB345 Lee et al, Scientific Reports 2016
Pan-axonal neurofilament SMI-312 1:500 Mouse brain 0.2 BioLegend SMI-312 Treweek et al, Nature Protocols 2015
Pan-cytokeratin (AE1/AE3) 1:100 Human tumor 3 eBiosciences 53-9003 Yang et al, Cell 2014
Parvalbumin (PV) 1:50-1:100 Mouse brain; human brain; moue pancreas; human pancreas 0.5-2 Abcam ab32895, ab11427, ab64555 Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014; Murray et al, Cell 2015; Costantini et al, Scientific Reports 2015; Yu et al, Scientific Reports 2017; Hsueh et al, Scientific Reports 2017
1:100-1:200 Mouse brain 0.25-1 Swant PVG-214, 235, PV27 Unal et al, J. Neurosci. 2015; Phillips et al, Scientific Reports 2016; Bastrup and Larsen, J. Neuro. Methods 2017
1:500 Mouse brain 0.5 Synaptic Systems 195004 Unal et al, J. Neurosci. 2015
1 Millipore MAB1572 Lee et al, Scientific Reports 2016
1:200 Mouse brain 0.1 Immunostar Sung et al, Scientific Reports 2016
1:100 Mouse brain 1 Sigma P3088 Bastrup and Larsen, J. Neuro. Methods 2017
Platelet-derived growth factor receptors (PDGFR) 1:500 Mouse brain 1 Becton Dickinson 558774 Lee et al, Scientific Reports 2016
Platelet-endothelial cell adhesion molecule 1 (PECAM1) 1:10 Mouse ovary Abcam ab28364 Feng et al, Scientific Reports 2017; Lu et al, Reproduction 2017
Podocin 1:50 Rat kidney 1-1.5 Sigma-Aldrich P0372 Unnersjo-Jess et al, Kidney International 2016
Polysialylated neuronal cell adhesion molecule (PSA-NCAM) 1:500 Mouse brain 1 Millipore MAB5324 Lee et al, Scientific Reports 2016
Porin 1:100 Mouse brain; human brain 0.25 Abcam ab14734 Phillips et al, Scientific Reports 2016
Postsynaptic density protein 95 (PSD-95) 1:50-1:100 Mouse brain 0.5-1 Abcam ab2723, ab12093 Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014; Murray et al, Cell 2015
1:500 Mouse brain 1 Invitrogen 51-6900 Lee et al, Scientific Reports 2016
1:1000 Mouse brain 1 Pierce MA1-045 Poguzhelskaya et al, Mol. Neurodeg. 2014
Pro-oxyphysin (PS38) 1:200 Rat brain 2 ATCC CRL-1950 Zheng and Rinaman, Brain Struct. Funct. 2016
Proliferating cell nuclear antigen (PCNA) 1:250 Mouse brain 1 Santa Cruz Biotechnology sc-56 Lee et al, Scientific Reports 2016
Protein gene product 9.5 (PGP9.5) 1:500 Mouse brain; human spinal cord 1 Upstate Biotechnology 06-570 Lee et al, Scientific Reports 2016
Red fluorescent protein (RFP) 1:300 Mouse brain 1 Abcam ab62341 Lee et al, Scientific Reports 2016
Regulated in development and DNA damage responses-1 (REDD1) 1:250 Mouse brain 1 ProteinTech Group 10638-1-AP Lee et al, Scientific Reports 2016
S100 1:500 Mouse brain 1 Sigma s2532 Lee et al, Scientific Reports 2016
Serotonin 1:400 Mouse intestine Immunostar 20080 Neckel et al, Scientific Reports 2016
1:100 Mouse spinal cord Millipore ab938 Liang et al, Molecular Pain 2015
Smooth muscle actin 1:100 Mouse kidney Santa Cruz Biotechnology sc-53142 Epp et al, eNeuro 2015
Mouse intestine; mouse brain; human brain 0.25-1 Dako M0851 Neckel et al, Scientific Reports 2016; Phillips et al, Scientific Reports 2016
Smooth muscle protein 22-alpha (SM22 alpha) 1:300 Mouse brain 1 Abcam ab14106 Lee et al, Scientific Reports 2016
Somatostatin 1:100-1:200 Mouse brain; human brain; mouse pancreas; human pancreas 0.5-1+ Peninsula T-4103 Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014; Hsueh et al, Scientific Reports 2017
1:100 Mouse brain 1 Millipore MAB354 Murray et al, Cell 2015
1:500 Human pancreas 2 Santa Cruz Biotechnology sc-7819 Treweek et al, Nature Protocols 2015
Special AT-rich sequence-binding protein-2 (SATB2) 1:500 Mouse brain 1 Abcam ab34735 Lee et al, Scientific Reports 2016
Succinate dehydrogenase complex, subunit A (SDHA) 1:100 Mouse brain; human brain 0.25 Abcam ab14715 Phillips et al, Scientific Reports 2016
Synapsin-1 1:50-1:200 Mouse brain 0.5-1 Abcam ab64581 Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014
1:100 Mouse brain 1 Cell Signaling Technology 5297 Murray et al, Cell 2015
Synaptophysin 1:100 Mouse brain 1 Abcam ab8049 Murray et al, Cell 2015
Tau 1 1:50 Mouse brain 3 Millipore MAB3420 Lee et al, BMC Develop. Bio. 2014
Tau B19 Human brain 0.5 Covance Ando et al, Acta Neuropath. 2014
Tight junction protein-1 (ZO-1) 1:100 Mouse intestine; human intestine Thermo Fisher 33-9100 Neckel et al, Scientific Reports 2016
Trombosphondin 4 1:500 Mouse brain 1 R&D Systems AF3074 Lee et al, Scientific Reports 2016
Tropomyosin receptor kinase A (TrkA) 1:500 Mouse brain; rat embryo 1 R&D Systems AF1056 Lee et al, Scientific Reports 2016
Tyrosine hydroxylase (TH) 1:50-1:100 Mouse brain; human brain; zebrafish brain; mouse ovary 0.5-5 Abcam ab113, ab51191, ab134461, ab75875, ab41528, ab112, ab76442 Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014; Murray et al, Cell 2015; Feng et al, Scientific Reports 2017; Lu et al, Reproduction 2017; Romanov et al, Nature Neurosci. 2017
Human brain; mouse spleen 3 Millipore AB152 Liu et al, Neuropathol. Appl. Neurobiol. 2016; Lai et al, PLoS One 2016; Murray et al, PLoS One 2017
1:300-1:600 Mouse brain; rat brain; zebrafish brain; Xenopus brain 1-2+ Millipore AB152, MAB318 Lee et al, Scientific Reports 2016; Zheng and Rinaman, Brain Struct. Funct. 2016; Xavier et al, J. Comparative Neurology 2017
1:400 Mouse brain 1 Aves TYH Yang et al, Cell 2014
Mouse intestine Immunostar 22941 Neckel et al, Scientific Reports 2016
1:100 Mouse brain 1 Covance MMS-5210, PRB-515P Murray et al, Cell 2015
1:250 Mouse brain 1 Santa Cruz Biotechnology sc-14007 Lee et al, Scientific Reports 2016
1:50 Mouse brain 1 Calbiochem Nordstrom et al, Neurobio. Disease 2015
Vascular endothelial growth factor (VEGF) 1:50 Mouse ovary Abcam ab46154 Feng et al, Scientific Reports 2017
Vasoactive intestinal peptide (VIP) 1:100 Mouse brain 1 Immunostar 20077 Murray et al, Cell 2015
Vesicular glutamate transporter-1 (VGlut1) 1:500 Mouse brain 1 Synaptic Systems 135311 Lee et al, Scientific Reports 2016
Vimentin 1:300 Mouse brain 1 Millipore AB1620 Lee et al, Scientific Reports 2016
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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