The brain sections on slides were dried at room temperature (RT) for 20 min. The slides were washed twice with PBSTX (1× PBS/0.3% Triton X-100) for 10 min. After incubation with 10 mM sodium citrate for 15 min at 85°C, they were washed with 1 × PBS twice for 5 min and incubated with diluted primary antibody in PBSTX at 4°C overnight. The next day, the slides were washed three times with PBSTX for 10 min at RT, incubated with secondary antibody, and washed three times with 1× PBS for 5 min at RT. Images were obtained using an LSM 880 laser scanning confocal microscope (Carl Zeiss AG, Oberkochen, Germany). The primary antibodies are as follows: mouse anti-GFAP (1:500, ab154474, Abcam, Cambridge, United Kingdom), mouse anti-PCNA (1:500, M0879, Dako, Agilent, CA, United States), rabbit anti-S100β (1:500, Z0311, Dako, Agilent), mouse anti-acetylated tubulin (1:250, T6793, Sigma-Aldrich, MO, United States), and rabbit anti-phospho-β-catenin (1:100, D2F1, Cell Signaling Technology, MA, United States). The secondary antibodies are as follows: rhodamine (TRITC) AffiniPure donkey anti-rabbit IgG (1:200, 711-025-152, Jackson Immunoresearch Laboratories, PA, United States) and Cy5 AffiniPure donkey anti-rat IgG (H+L) (1:400, 712-175-150, Jackson Immunoresearch Laboratories). Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI; 4083S, Cell Signaling Technology).
Z0311
Manufactured by Agilent Technologies
Sourced in United Kingdom, United States, Denmark
The Z0311 is a laboratory equipment product from Agilent Technologies. It is designed for performing specific scientific measurements and analyses in a controlled laboratory environment. The core function of the Z0311 is to provide accurate and reliable data for research or testing purposes.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab15580, by Abcam (2 mentions)
Donkey serum, by Jackson ImmunoResearch (2 mentions)
Flouromount g, by Southern Biotech (2 mentions)
Goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Mouse anti gfap, by Abcam (1 mentions)
M0879, by Agilent Technologies (1 mentions)
T6793, by Merck Group (1 mentions)
Ab154474, by Abcam (1 mentions)
Lsm 880 confocal laser scanning microscope, by Zeiss (1 mentions)
Tritc affinipure donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Aperio imagescope, by Leica (1 mentions)
Ga626, by Agilent Technologies (1 mentions)
Ab6992, by Abcam (1 mentions)
Ab75810, by Abcam (1 mentions)
Ab4680, by Abcam (1 mentions)
Ab5320, by Merck Group (1 mentions)
Mitomycin, by Merck Group (1 mentions)
N6 2 o dibutyryladenosine 3 5 cyclic monophosphate sodium salt dbcamp, by Merck Group (1 mentions)
Mca341r, by Bio-Rad (1 mentions)
32 protocols using z0311
1
Immunohistochemical Analysis of Brain Sections
2
Quantifying Melanoma Tumor Characteristics
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Upon sacrifice, the tumors were excised and processed for (immuno) histology. Tissue sections (5 μm thick) cut perpendicular to the surface and showing all layers of skin and tumor (top to bottom) were stained with H&E and immunostained for S100 (1:500 Z-0311; Dako–Agilent Technologies, Santa Clara, CA, USA) and Ki67 (1:500 GA626; Dako–Agilent) expression as markers of the presence of melanoma cells [31 ], and cell viability and proliferation [32 (link)], respectively. WARP-Red chromogen, an alkaline phosphatase system, was used to distinguish between the positive-stained cells and melanin pigmentation. Two sections from each tumor were stained and digitized (Aperio ImageScope®; Leica, Canada) and the images were processed using commercial software (HALO®, Indica Labs) to measure the tumor area and total tumor cell count and to quantify the specific protein-expression levels. Immunostaining was considered negative when <10% of tumor cells showed positivity. The S100-stained sections were used to measure the largest tumor thickness, with three measurements being performed in each section.
3
Neurotoxin and Antibody Protocol
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α-LTx (LSP-130) and µ-conotoxin GIIIB (C-270) were purchased from Alomone. α-LTx purity was checked by SDS–PAGE, and its neurotoxicity by ex vivo mouse nerve-hemidiaphragm preparations, as previously described [28 (link)]. BoNT/A (Xeomin) was from Merz. All other reagents were from Sigma unless stated otherwise.
Antibodies and fluorescent conjugates with relative dilutions: α-BTx AlexaFluor555 (B35451 Thermo Fisher, 1:200), anti-Ctgf neutralizing antibody (70R-CR023 Fitzgerald, 2 µg/40 µl), anti-Ctgf for immunostaining (ab6992 Abcam, 1:200), anti-S100 (Z0311 Dako, 1:400), anti-GAP43 (ab75810 Abcam, 1:200), anti-NF (ab4680 Abcam, 1:800), anti-VAMP1 [29 (link)], anti-SNAP25 (ab24737 Abcam, 1:200), anti-SNAP-25 BoNT/A-cleaved [30 (link)], anti-syntaxin 1A/1B [31 (link)], anti-YAP (13008S, Cell Signaling, 1:200). Secondary AlexaFluor-conjugated antibodies (1:200) were from Thermo Fisher. A list of antibodies and the relative description is provided as Additional file9 .
Antibodies and fluorescent conjugates with relative dilutions: α-BTx AlexaFluor555 (B35451 Thermo Fisher, 1:200), anti-Ctgf neutralizing antibody (70R-CR023 Fitzgerald, 2 µg/40 µl), anti-Ctgf for immunostaining (ab6992 Abcam, 1:200), anti-S100 (Z0311 Dako, 1:400), anti-GAP43 (ab75810 Abcam, 1:200), anti-NF (ab4680 Abcam, 1:800), anti-VAMP1 [29 (link)], anti-SNAP25 (ab24737 Abcam, 1:200), anti-SNAP-25 BoNT/A-cleaved [30 (link)], anti-syntaxin 1A/1B [31 (link)], anti-YAP (13008S, Cell Signaling, 1:200). Secondary AlexaFluor-conjugated antibodies (1:200) were from Thermo Fisher. A list of antibodies and the relative description is provided as Additional file
4
Immunochemical Identification of CNS Cell Types
5
S100 Immunohistochemistry in Paraffin Sections
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Formalin-fixed tissue was embedded in paraffin and 4 µM sections were cut, dewaxed, and rehydrated. Sections were depigmented with 10% H2O2 in 0.05 M PBS (pH 7.4) for 1.5 h at 55 °C before antigen retrieval. Antigen retrieval was performed using citrate buffer pH 6 (Vector Laboratories, H-3300) and PickCell electric pressure cooker. Next, slides were rinsed with PBS (3 × 5 min), followed by blocking of peroxidase for 10 min with 3% H2O2 in methanol and again rinsed with PBS (3 × 5 min). Blocking was performed with 5% goat serum in 1% BSA in PBS for 30 min. Slides were incubated with primary anti-S100 antibody (1/20000, DAKO, Z0311) in 1% BSA in PBS overnight at 4 °C and secondary goat anti-rabbit Ab-biotin (1/500, DAKO, E0432) in blocking buffer. Rinsing steps were always performed with PBS (3 × 5 min). Finally, slides were incubated with ABC (Vector Laboratories, PK-7100) for 30 min and incubated with DAB until specific staining appeared. Reaction was stopped with tap water and slides were mounted with xylene-based mounting medium. Lymph node area, S100 area, and melanin area (the latter visible on H/E stainings) were quantified using QuPath. Outliers (values higher/lower than 2 x SEM) were removed from the analysis.
6
Immunohistochemical Analysis of Mouse Melanomas
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Mouse melanomas were collected, rinsed in cold PBS and fixed in 4% PFA at 4 °C O/N. Samples were dehydrated, embedded in paraffin wax and sectioned into 5-μm-thick transverse sections. Paraffin-embedded sections were stained with haematoxylin and eosin, and examined by light microscopy. For immunostaining, sections were deparaffinized, rinsed in Tween Buffer saline (TBS; Tris 20mM pH 7,6, NaCl 150mM and Tween 20 0.1%), boiled for 20 min in 10 mM sodium citrate and treated overnight at 4 °C in TBST (TBS/0.1% Tween-20) containing 5% normal goat serum with antibodies against S100 (Dako, Z0311, dilution 1/100). AEC (Sigma-Aldrich, A6926) was used to reveal bound antibody according to the manufacturer's instructions. All sections were counterstained with haematoxylin. Ki-67 (Nova-Costra, NCL-Ki67p) and gp84 (ECAD antibody) antibodies were both produced in rabbit.
7
Immunocytochemistry of SC and Neuron Co-culture
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Primary rat and mice SCs and human iPSC-derived sensory neurons–SCs co-culture were fixed in 4% PFA for 10 min at RT. Primary cells were then permeabilized with 0.2% Triton X-100 for 5 min at RT, whereas the co-cultures were permeabilized with cold MeOH for 20 min on ice. Cells were incubated with primary antibody in blocking buffer (1% BSA in PBS) overnight at 4°C, followed by incubation with secondary antibodies in blocking buffer for 2.5 h at RT. Primary antibodies used in immunocytochemistry were monoclonal αms RalA (610222; BD Biosciences), 1 to 50; αrb S100 (Z0311; DAKO), 1 to 300; αrb Ki67 (ab16667; Abcam), 1 to 500; αck GFP (ab13970; Abcam), 1 to 200; αgt Talin (sc-7534; Santa Cruz Biotechnology), 1 to 50; αms Myc (2276s; Cell Signaling Technology), 1 to 500; αms neurofilament heavy chain (ab7795; Abcam), 1 to 10,000; αrt MBP (ab7349; Abcam), 1 to 400; and αrb S100 (GA504; DAKO), 1 to 300.
8
Immunofluorescence Staining of Brain Sections
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Brains were processed through perfusion/fixation, as above. Serial 30 μm, free-floating coronal sections representing 1/6th of each brain were permeabilized in Tris-buffered saline containing Triton X-100 (TBST; 50 mM Tris with 0.9% NaCl, and 0.5% Triton X-100), incubated with 0.3% H2O2, and blocked with 10% horse serum/TBST. Primary antibodies were incubated in 1% horse serum/TBST for 48 h. Primary antibodies: glial fibrillary acidic protein (GFAP; 1:500, anti-rabbit, Dako Z033429), sex-determining region box 2 (Sox2; 1:100, anti-goat, Santa Cruz Biotechnology, sc-17320), Ki67 (1:500, Abcam ab15580), brain lipid binding protein (BLBP; 1:300, EMD Millipore, ABN14), doublecortin (DCX; 1:50, Santa Cruz Biotechnology, sc-8066, or 1:200 Abcam ab18723), FGF-23 (1:30, R&D Systems, MAB26291; 1:100, Mybiosource MBS2003657), cleaved caspase 3 (1:1000, Cell Signaling 9664), or S100β (1:400, Dako, Z0311). Primary antibody binding was visualized after incubation in fluorescently labeled secondary antibody (Life Technologies). Nuclei were labeled with 4’,6-diamidino-2-phenylindole (DAPI; Life Technologies) and mounted in Prolong Gold anti-fade mounting media (Life Technologies).
9
Multiplex Immunohistochemistry of Tumor Vasculature
10
Hepatic Vagal Nerve Immunohistochemistry Protocol
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For hepatic vagal nerve immunohistochemistry, the oesophagus and vagal trunks were removed and fixed with 10% formalin and embedded in paraffin. In addition, splanchnic nerve was removed and fixed with 10% formalin and embedded in paraffin. Tissue sections were stained with haematoxylin–eosin. The streptavidin–biotin method was performed using a Histofine SAB-PO kit (Nichirei, Tokyo, Japan) and antibodies against calcitonin gene-related peptide (AB5920), tyrosine hydroxylase (AB152; Chemicon International, Temecula, CA, USA) and S-100 protein (Z0311; DAKO, Glostrup, Denmark). The antigen–antibody complex was visualized with 3,3'-diaminobenzidine and counterstained with haematoxylin3 (link)4 (link)35 (link).
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