Immunohistochemical stainings were performed as described in19 (link). Primary antibodies used are anti-Troponin T cardiac isoform (Thermo Scientific); anti-CD45, α-SMA, anti-Cytokeratin 10 and anti-Ki 67 (all from Abcam). For detection of binding of the primary antibody, the Ultra-Sensitive ABC Peroxidase rabbit/mouse IgG staining kit (Thermo Scientific, Waltham, MA, USA) and the ImmPACT VIP-Peroxidase substrate kit (Vector, Burlingame, CA, USA) were used according to the respective manufacturer’s protocols. Nuclei were counterstained with hematoxylin and slides were dehydrated, cleared in xylene, and mounted with Eukitt (Sigma Aldrich, USA).
Anti cd45
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Germany
Anti-CD45 is a laboratory equipment product designed for the detection and analysis of CD45, a transmembrane protein expressed on the surface of most hematopoietic cells. It is a key component used in various cell identification and separation techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd68, by Abcam (7 mentions)
Anti ki67, by Abcam (6 mentions)
Anti cd11b, by Abcam (5 mentions)
Anti cd31, by Abcam (5 mentions)
Anti cd105, by Abcam (4 mentions)
Anti cd34, by Abcam (3 mentions)
Anti cd44, by Cell Signaling Technology (3 mentions)
Anti epcam, by Abcam (3 mentions)
Anti cd4, by Abcam (3 mentions)
Anti f4 80, by Abcam (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Anti foxp3, by Abcam (2 mentions)
Moflo xdp flow cytometer, by Beckman Coulter (2 mentions)
Anti cd19, by Abcam (2 mentions)
Kaluza software, by Beckman Coulter (2 mentions)
Anti cd73, by Abcam (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
50 protocols using anti cd45
1
Immunohistochemical Staining Protocol
2
Immunofluorescence Staining and Quantification
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Histological analysis was performed as previously described (Zhao et al., 2019 (link); Dong et al., 2020 (link); Li et al., 2020b (link)). For immunofluorescence staining, antigen retrieval was performed by boiling the slides in sodium citrate (pH 6.0) for 1 min. The slides were washed 2 × 5 min in TBS plus 0.025% Triton X-100 with gentle agitation, blocked with 5% BSA, and incubated with anti-CD45 (Abcam, 1:200) overnight. After several washes with PBS, the slides were incubated with FITC-labeled secondary antibodies (Jackson, 1:200) for 30 min. DAPI (Sigma) was added and incubated with cells for 5 min prior to observation. Immunofluorescence was visualized on a confocal microscope (LSM 710, Zeiss). For quantification, stain-positive cells were counted on each slide and normalized to the control group which is arbitrarily set as 1. Images were quantified with ∼10 fields counted per mouse.
3
Immunofluorescence Staining of Cells
4
Histological Analysis of Cardiac Remodeling
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Four weeks after the AB operation, the mouse heart was removed, fixed with 10% paraformaldehyde, embedded in paraffin, and sectioned (5 μM thickness). Hematoxylin and eosin (HE) staining was used to evaluate the area of cardiomyocytes. The collagen deposition fraction was evaluated by picric acid red (PSR) staining. The myocardial cell area (at least 200 per group) and left ventricular collagen fraction were calculated by a quantitative digital image analysis system (Image-Pro Plus, IPP, version 6.0). Heart sections were incubated with anti-CD45 (Abcam, 1 : 100 dilution) or anti-CD68 (Abcam, 1 : 100 dilution) antibodies and then crosslinked with anti-rabbit horseradish peroxidase (HRP) reagent (Gene-tech, Shanghai, China). Finally, the DAB substrate kit (Gene-Tech, Shanghai, China) was used for coloration. A fluorescence microscope was utilized to acquire images and count CD45+CD68+ cells (10 fields for each heart).
5
Isolation of Murine Cancer-Associated Fibroblasts
6
Immunohistochemical and Immunofluorescent Analysis of RM-1 Tumors
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Formalin fixed and paraffin embedded RM-1 mouse tumors were subjected to immunohistochemistry (IHC) using Dako Autostainer Plus En VisionTM + Kit (Dako, Glostrup, Denmark) and stained with the antibodies anti-pSTAT3-T705 (ab76315 Abcam, Cambridge, UK), anti-pSTAT3-S727 (#9143 Cell Signaling Technology, Danvers, MA, USA), anti-FOXP3 (ab54501 Abcam) and anti-CD45 (ab25386 Abcam). The immunostainings were analyzed using Halo image analysis software (Indica Laboratories, Albuquerque, NM, USA).
For immunofluorescent staining, the RM-1 tumors were stained using the Opal Multiplex immunofluorescence assay (Akoya Biosciences) and imaged with Mantra multispectral image system (Akoya Biosciences). Antibodies used were anti-CD3E (LS-C343957 LSBio), anti-CTLA-4 (ab 237,712 Abcam) and anti-pSTAT3-S727 (#9143 Cell Signaling Technology).
For immunofluorescent staining, the RM-1 tumors were stained using the Opal Multiplex immunofluorescence assay (Akoya Biosciences) and imaged with Mantra multispectral image system (Akoya Biosciences). Antibodies used were anti-CD3E (LS-C343957 LSBio), anti-CTLA-4 (ab 237,712 Abcam) and anti-pSTAT3-S727 (#9143 Cell Signaling Technology).
7
Western Blot Analysis of Penile Tissue
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Penile tissue was homogenized in RIPA lysis buffer. The tissue was dispersed using ultrasound, centrifuged and the supernatant collected. Protein concentrations were determined using the BCA assay kit (Beyotime). Equal amounts (30 mg of protein extract/lane) of protein from each sample were electrophoretically separated on 12% SDS‐PAGE gels and transferred to PVDF membranes for analysis. After blocking for 1.5 h using bovine serum albumin at room temperature, the membranes were incubated with antibodies overnight at 4°C. Primary antibodies included polyclonal rabbit anti‐CD45 (1:1000, Abcam), anti‐CD11b (1:1000, Abcam), anti‐Nrf2 (1:1000, Abcam), anti‐HO‐1 (1:1000, Abcam), anti‐NF‐κB(p65) (1:1000, Abcam), anti‐P38 (1:1000, Abcam), anti‐p‐P38 (1:1000, Abcam), anti‐eNOS (1:1000, Abcam), anti‐p‐eNOS (1:1000, Abcam) and anti‐β‐actin (1:8000, Santa Cruz Biotechnology). The secondary antibody was horseradish peroxidase‐conjugated to mouse anti‐rabbit/mouse IgG (1:5000, Santa Cruz Biotechnology). Immunoblots were developed by Enhanced Chemiluminescence (ECL). Protein band densities were quantified using Image J software (NIH, Scion Corpomiceion) and were normalized to β‐actin. Final data were expressed as a per cent of controls.
8
Quantifying Immune Cell Populations
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Immunohistochemical staining of F4/80, CD45 were performed adhering to instructions provided by UltraSensitive™ SP Kit (KIT-9710, Maxim Bio, Fuzhou, China). The primary antibodies used were as follows: anti-CD45 (1: 8000, Abcam), and anti-F4/80 (GB11027, 1: 1000, Servicebio, Wuhan, China). Sections were incubated with non-immune serum instead of the primary antibody that was designated as NCs. Images were analyzed with Image-Pro Plus 5.1. software (Media Cybernetics, Inc., Rockville, MD, USA). Immunoactivity of F4/80 and CD45 were quantified with the percent of positive cells [46 (link)].
9
Immunocytochemistry Analysis of Adherent Cells
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Monolayer and single colony-derived adherent cells (at the third passage in culture) were analyzed by immunocytochemistry. Cytospin preparations and growing cells in 6-well culture plate were fixed in 4 % paraformaldehyde for 20 min at 4 °C, washed three times with PBS, and then incubated with 3 % hydrogen-peroxide (H2O2; Sigma-Aldrich) at room temperature for 30 min. The cells were permeabilized and pre-incubated with blocking solution (containing 2 % goat serum, 0.3 % Triton X-100, and 0.1 %BSA in PBS) for 30 min at room temperature, and then blocked with 5 % normal goat serum at room temperature for 30 min. Washed slides were separately incubated with anti-CD44 (1:50, Abcam, USA), anti-CD29 (1:50, Abcam, USA) and anti-CD45 (1:50, Abcam, USA) primary antibody at 4 °C overnight. After washing with PBS, Alexa Fluor 488 anti-goat IgG (1:200; Molecular Probes, Carlsbad, CA, USA) was incubated for 1 h at 37 °C. Nuclear staining was performed by treatment with 4′, 6-diamidino-2-phenylindole (DAPI, 1:20,000; Molecular Probes, Carlsbad, CA, USA) for 5 min. Slices were then mounted and observed with a florescent microscope (Leica, Solms, Germany).
10
Investigating p38 MAPK Signaling Pathways
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The anti-p38α and p-p38 used in western blot (WB) or immunoprecipitation (IP) or immunofluorescence (IF), and anti-p38β, anti-p38γ, anti-p38δ used in WB or IP, and anti-β-actin, anti-MKK-3, anti-MKK-4, anti-MKK-6 used in WB were purchased from CST. The anti-p38βand CD206 were purchased from ST, and anti-p38γ and anti-p38δ were from RD, the anti-CD68, anti-CD45, anti-CD24, anti-CD11b, anti-iNOS and anti-Arginase-1 were obtained from Abcam were used in IF. Anti-IFNγ and anti-GM-CSF uesd in neutralising assay were purchased from Abcam. BDTM Flow Cytometric Bead (CBA) Array flex set (Human IFNγ GM-CSF, TNF-α, IL-1β, MCP-1 flex set) was purchased from BD Biosciences. Plasmids short hairpin RNA for p38α, p38β p38γ and p38δ in a lentiviral GV118 vector were obtained from Genechem (Genechem Shanghai, China). IL-1β was purchased from PeproTech.
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