For the detection of hypoxia, mice were treated with 60 mg/kg of pimonidazole HCl (HP1-200Kit; Hypoxyprobe, USA) 30 min prior to sacrifice. Colon samples were fixed in 4% paraformaldehyde, and paraffin-embedded tissues were probed with mouse anti-pimonidazole monoclonal antibody (1:100; Hypoxyprobe) and then stained with Cy-3-conjugated goat anti-mouse antibody (1:50; SA00009-1, Proteintech), and then counterstained with DAPI using anti-fading mountant(S2110, Solarbio, China). Representative images were obtained using a Nikon fluorescent microscope (TE2000, Nikon, Tokyo, Japan).
Sa00009 1
Manufactured by Proteintech
Sourced in United States, China
The SA00009-1 is a laboratory equipment product designed for general laboratory use. It serves a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Sa00003 2, by Proteintech (5 mentions)
Dapi fluoromount g, by Southern Biotech (3 mentions)
Sa00013 2, by Proteintech (2 mentions)
Ab4066, by Abcam (2 mentions)
Pa5 76664, by Thermo Fisher Scientific (2 mentions)
Axio imager m2, by Zeiss (2 mentions)
Ab150077, by Abcam (2 mentions)
Mouse anti pimonidazole monoclonal antibody, by Hypoxyprobe (1 mentions)
S2110, by Solarbio (1 mentions)
Fluorescent microscope, by Nikon (1 mentions)
Te2000, by Nikon (1 mentions)
Ctr4000b, by Leica camera (1 mentions)
Rabbit polyclonal, by Proteintech (1 mentions)
P0106, by Beyotime (1 mentions)
Abs137082, by Absin (1 mentions)
Sa00009 3, by Proteintech (1 mentions)
Nb100 2828, by Novus Biologicals (1 mentions)
L9393, by Merck Group (1 mentions)
Mca2077s, by Bio-Rad (1 mentions)
Evos m5000 microscope, by Thermo Fisher Scientific (1 mentions)
14 protocols using sa00009 1
1
Hypoxia Detection in Murine Colon
2
Immunofluorescence Assay for Cartilage Markers
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The treated cells were fixed with formaldehyde for 10mins and incubated in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the primary anti-COL2A1 (1:50, Santacruz biotechnology, sc-52658) and ACAN (1:200, proteintech, 13880-1-AP) antibody overnight at 4°C. The secondary antibody (green and red) was goat anti rabbit and mouse (proteintech, SA00003-2 and proteintech, SA00009-1) Ig G(H+L) which were used at a dilution of 1 to 50 for 1h. DAPI was used to stain the cell nuclei(blue) and its concentration was 1.43μM. The cells were observed by fluorescence microscope (CTR4000B, Leica). Experiments were repeated three times independently.
3
Immunofluorescence Staining of CD14
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The obtained tissue sections (10 μm sheet thickness) were also co-incubated with primary CD14 (1: 200) antibody (Rabbit Polyclonal, proteintech, China) overnight at a 4 °C refrigerator. After washing with detergent (P0106, Beyotime, China) three times for 5 min, the samples were co-incubated again with Cy3 secondary antibody (1: 50, SA00009-1, proteintech, China) and dark atmosphere overnight at a 4 °C refrigerator. The samples were observed by a fluorescence microscope.
4
Immunohistochemical Analysis of Brown and White Adipose Tissue
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The treated-side SS (n = 5 per group) cryosection slides were fixed in 4% paraformaldehyde for 30 minutes, rinsed in PBS, placed in 0.1 M glycine (diluted in PBS; Fisher Scientific) for 30 minutes, and washed again in PBS. They were then covered with blocking solution (0.2% Triton X-100; 2% bovine serum albumin in PBS) for 1 hour at room temperature. Primary antibodies against UCP1 (an indicator of BAT activation or white fat browning) (diluted 1:100; NB100-2828; Novus Biologicals), tyrosine hydroxylase (TH) (an indicator of activation of sympathetic nerve fibers) (diluted 1:500; 66334-1-Ig; Proteintech), laminin (diluted 1:500; L9393; Sigma-Aldrich), and perilipin (diluted 1:500; abs137082; Absin) were diluted in a block mix and added to the sections for overnight incubation at 4°C. After a PBS rinse, the sections were incubated with a mixture containing FITC-conjugated (diluted 1:500; SA00003-8, SA00003-2; Proteintech) and Cy3-conjugated (diluted 1:500; SA00009-3, SA00009-1; Proteintech) secondary antibodies at room temperature for 120 minutes. After a PBS rinse for 2 × 10 minutes, the slides were covered with DAPI containing antifade mounting medium.
5
Immunofluorescence Analysis of Urogenital Tissues
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The embedded urogenital tissues were sliced continuously with the thickness of 10 μm in the frozen slicer. Then, the sections were fixed in 4% paraformaldehyde for 15 min, permeabilized in PBS containing 1% Triton X-100 at room temperature for 10 min, and blocked at 37 °C in PBS containing 5% BSA for 1 h. The primary antibody solution was diluted with 1% PBS, and DMRT1 antibody (1:100, AbClone, WG-03818, Wuhan, China), SOX9 antibody (1:100, Abcam, EPR12755, Cambridge, UK), FOXL2 antibody (1:200, Novus, NB100-1277s), and CYP19A1 antibody (1:50, Bio-Rad, MCA2077S) were added and incubated overnight at 4 °C. Secondary antibody goat anti-rabbit IgG (AbClone, AS007), goat anti-mouse (ProteinTech, SA00009-1, Wuhan, China), or rabbit anti-goat (ProteinTech, SA00009-4) was diluted with PBS at a ratio of 1:200, incubated at 37 °C for 1 h in dark, and washed 5 times with 1% PBS. Finally, sections were stained with 1 mg/mL of 4′, 6-diamidino-2-phenylindole (DAPI) for 10 min, sealed with an anti-fluorescence quenching agent, and imaged under a microscope equipped with fluorescence optics.
6
Immunophenotyping of RM cells and AGC tissue
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RM cells immunofluorescence assay was performed in 24-well plates; the AGC tissue was first frozen and sliced, then used for immunofluorescence assay. The cells and tissues were fixed, permeabilized, blocked, and incubated overnight with primary antibodies: CD90 (1:300, proteintech, USA, 66766-1-lg), CD73 (1:1000, proteintech, USA, 12231-1-AP), Nestin (1:300, BIOSS, China, bs-0008R), CD34 (1:300, proteintech, USA, 14486-1-AP), and Prrx1 (1:200, Absin, China, abs134576). The cells were then incubated with secondary antibodies (1:50, proteintech, USA, SA00009-1, SA00009-2, SA00013-2) for 2 h, respectively. Finally, the cells and tissues were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Images were obtained and analyzed using EVOS M5000 microscope (Thermo Fisher, USA).
7
Immunofluorescence Staining of Neuronal Proteins
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Cells were seeded in a 24‐well plate, incubated overnight and fixed with 4% paraformaldehyde for 15 minutes. Cells were permeabilized with 0.1% Triton at room temperature for 15 minutes and then blocked with 5% FBS for 15 minutes. Next, cells were incubated at 4°C overnight with anti‐NETO1 (1 μg/106 cells) or anti‐KIF2A (2.5 μg/106 cells), anti‐β‐tubulin (1:50) and anti‐F‐actin (1:1000). After PBS washing, the cells were then incubated with goat anti‐rabbit FITC (1:200, SA00003‐2; Proteintech, Chicago, Illinois, USA) or goat antimouse CY3 (1:200, SA00009‐1; Proteintech, Chicago, Illinois, USA) for 1 hour at room temperature. Finally, nuclei were labelled with Hoechst dye for 15 minutes. Images were acquired by fluorescent microscope.
8
Antibody Validation for Immunological Assays
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The following primary antibodies were used in this study: mouse monoclonal anti-FLAG (M20008; Abmart), mouse monoclonal anti-β-actin (66009-1-lg; Proteintech), rabbit polyclonal anti-TMEM53 (HPA021134; Sigma-Aldrich), mouse monoclonal anti-dsRNA J2 (J2-1702; Scicons), rabbit polyclonal anti-STAT1 (10144-2-AP; Proteintech), mouse monoclonal anti-HA (TA180128; OriGene), rabbit polyclonal anti-HA (H6908; Sigma-Aldrich), and mouse anti-IgG (A7028, Beyotime). Anti-S-tag and anti-NP monoclonal antibodies were prepared in-house. We used the following secondary antibodies: horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (SA00001-1, Proteintech), HRP-conjugated goat anti-rabbit IgG (SA00001-2, Proteintech), goat anti-mouse IgG H&L DyLight 488 (ab96879, Abcam), goat anti-rabbit IgG H&L Cy3 (ab6939, Abcam), and goat anti-mouse IgG H&L Cy3 (SA00009-1, Proteintech). Universal type I interferon (IFN) (11200, PBL) was acquired from PBL Assay Science.
9
Immunofluorescence Staining of Cultured Schwann Cells
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Cultured Schwann cells were fixed with 4% paraformaldehyde and exposed to primary antibodies rabbit anti-Btc (1:100, PA5-76664, Invitrogen) and mouse anti-S100 (1:100, ab4066, Abcam, Cambridge, MA, USA) followed by reaction with secondary antibodies Alexa Fluor 488-conjugated anti-rabbit (1:400, ab150077, Abcam) and Cy3-conjugated anti-Mouse (1:400, SA00009-1, Proteintech, Rosemont, IL). Cell nuclei were stained with DAPI Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Images were captured using Zeiss Axio Imager M2 (Carl Zeiss Microscopy GmbH, Jena, Germany).
10
Immunohistochemistry Analysis of Bone Tissues
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Bone tissues from each group of rats were processed for routine paraffin embedding. The slices were incubated at 3% H2O2/PBS at room temperature for 10 min to quench endogenous peroxidase activity. After rinsing with PBS, the slices were immersed in antigen repair solution. Blocking was performed with 5% BSA. Then, anti-CD31 antibody (1:200; Abcam, ab24590, UK), anti-endomucin (Emcn) antibody (1:200, Abbkine, ABP55560, China), and anti-Osterix antibody (1:1000, Abcam, ab209484) were added. The slices were incubated overnight at 4 °C, followed by washing with PBS. Corresponding Goat Anti-Rabbit (1:200, Proteintech, SA00003-2, USA) and Anti-Mouse (1:200, Proteintech, SA00009-1) secondary antibodies were added for 1 h. At last, the slices were stained with Hochest (Solarbio, Beijing, China), dehydrated, mounted, and observed under a microscope.
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