Paraffin-embedded rat kidney sections (4 μm thickness) were prepared by a routine procedure [17 (link)]. Sections were stained with periodic acid-Schiff reagent by standard protocol. Kidney sections were also subjected to Masson’s Trichrome staining for assessing collagen deposition and fibrotic lesions. Semi-quantitative determination of renal fibrosis score was carried out by previously reported methods [32 (link)]. Briefly, Masson trichrome-stained kidney sections were graded for the presence of interstitial fibrosis according to the following scale: 0, no evidence of interstitial fibrosis; 1, <25% involvement; 2, 25to 50% involvement, and 3, >50% involvement. The scale for each rat was reported as the mean of 10 random high-power (X400) fields per section [32 (link)]. Immunohistochemical staining was performed using established protocol, as described previously [9 (link)]. Antibodies used were as follow: rabbit polyclonal anti-α-smooth muscle actin antibody (ab5694), rabbit monoclonal anti-β-catenin antibody (ab32572), rabbit monoclonal anti-CD3 antibody (ab16669; Abcam, Cambridge, MA), mouse monoclonal anti-CD68 antibody (MCA341GA; Serotec), goat polyclonal anti-AGT (sc-7419), goat polyclonal anti-renin (sc-27320, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-AT1 receptor (AB15552; Millipore, Billerica, MA).
Mca341ga
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
The MCA341GA is a lab equipment product manufactured by Bio-Rad. It serves as a core function for laboratory applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (4 mentions)
Sc 33560, by Santa Cruz Biotechnology (2 mentions)
Ab15323, by Abcam (2 mentions)
Ab64693, by Abcam (2 mentions)
Ab16669, by Abcam (1 mentions)
Ab15552, by Merck Group (1 mentions)
Ab5694, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Mab377, by Merck Group (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Anti phospho stat1 tyr701, by Cell Signaling Technology (1 mentions)
Tetramethylrhodamine isothiocyanate conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Fluorescein isothiocyanate conjugated, by Jackson ImmunoResearch (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Sodium citrate, by Merck Group (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Af2330, by R&D Systems (1 mentions)
Ac 15, by Merck Group (1 mentions)
Abc elite kit, by Vector Laboratories (1 mentions)
C2456, by Merck Group (1 mentions)
17 protocols using mca341ga
1
Histological Analysis of Rat Kidney Fibrosis
2
Immunohistochemical analysis of neuroinflammation
3
Immunofluorescence Staining for M1 and M2 Macrophages
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For immunofluorescence staining, tissue sections were double-stained with anti-CD68 (1:500; MCA341GA, Serotec, United Kingdom) and anti-inducible nitric oxide synthase (iNOS) (1:100; ab-15323, Abcam) for M1 macrophages, or anti-CD68 and anti-CD163 (1:100; sc-33560, Santa Cruz) for M2 macrophages. PDLSCs were stained with anti-LC3B (1:100, CST3868S, CST). Samples were incubated with primary antibodies at 4°C overnight. On the following day, the samples were incubated with fluorescein secondary antibody (1:200, Jackson Immuno Research Laboratories, West Grove, PA, United States). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Samples were observed with a laser scanning microscope (LSM 510, Zeiss), and the images were processed using LSM 5 Release 4.2 software. Three different slides from each sample (n = 6) were used for cell counting. Each slide or cell sample was measured for three times and the average positive cell numbers were calculated.
4
Immunofluorescence Staining for M1/M2 Macrophages
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Immunofluorescence staining was performed as previously described [39 (link)]. The sections were double stained with antibodies consisting anti-CD68 (1:600; MCA341GA, Serotec, UK), and anti-inducible nitric oxide synthase (iNOS) (1:100; ab-15323, Abcam), or anti-CD68 and anti-CD163 (1:100; sc-33560, Santa Cruz) to detect M1 or M2 macrophages. In addition, anti-phospho-STAT1 (Tyr701) (1:300; #9167, Cell Signaling) antibodies were used to detect the influence of H2S on the STAT1 signaling pathway.
The sections were then incubated with fluorescein isothiocyanate-conjugated or tetramethylrhodamine isothiocyanate-conjugated secondary antibody (1:200, Jackson Immuno Research Laboratories, West Grove, PA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Confocal microscopic images were acquired using a laser scanning microscope (LSM 510, Zeiss, Jena, Germany), and the images were processed using LSM 5 Release 4.2 software. The positively stained cells were counted in five different slides from each sample (N = 5–6). The final result came from the average of three tests.
The sections were then incubated with fluorescein isothiocyanate-conjugated or tetramethylrhodamine isothiocyanate-conjugated secondary antibody (1:200, Jackson Immuno Research Laboratories, West Grove, PA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Confocal microscopic images were acquired using a laser scanning microscope (LSM 510, Zeiss, Jena, Germany), and the images were processed using LSM 5 Release 4.2 software. The positively stained cells were counted in five different slides from each sample (N = 5–6). The final result came from the average of three tests.
5
Macrophage Localization in Aortic Wall
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Staining and immunohistochemical analysis were performed on the paraffin-embedded tissue samples from the aortic wall to determine morphological changes and localize the distribution of the accumulation of macrophages. Macrophages were identified using mouse anti rat–CD68 antibody (dilution 1/50, Serotec MCA341GA). Endogenous peroxidase activity was quenched and the antigen was retrieved by treating the paraffin-embedded sections with sodium citrate 10 mmol/L, pH 6 (Sigma-Aldrich). Immunostaining employed appropriate biotinylated secondary antibodies (1/200, Vector laboratories, Burlingam, CA), streptavidin–horseradish peroxidase (Vectastain ABC kit, Vector laboratories), and the 3-amino-9-ethylcarbazole substrate–chromogen system (Sigma-Aldrich) for visualization. Finally, the slides were mounted with Glycergel (DAKO Corporation) and analyzed under a light microscope. Negative controls were tested both by omitting the primary antibody, as well as replacing it with an unrelated primary antibody. Both sets of controls yielded negative results, as expected.
6
Immunoblot Analysis of Glycosylation
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Immunoblot analysis was performed as described previously (Kawahara et al., 2009 ). The blotted membraneswere incubated with one of the following antibodies: 9F5 (1 µg ml−1), ED1 (1 µg ml−1; MCA341GA; Serotec), rabbit anti‐Iba1 (1 µg ml−1;019‐19741; Wako), goat anti‐GPNMB (1 µg ml−1;AF2330; R&D Systems), rabbit anti‐furin (1:500; PA1‐062; Affinity BioReagents), goat anti‐CD40 (1:100; T‐20; Santa CruzBiotechnology), goat anti‐CD86 (1:500; 421340; Genezyme Techne), or mouse anti‐β‐actin (1:2,000; AC15; Sigma–Aldrich) antibodies.
To determine the amount of N‐linked glycosylation, the cell lysate from rat type 1 MG was first denatured in 1% sodium dodecyl sulfate (SDS) (5 min, 60°C). Deglycosylation was then performed with 60 U ml−1 peptide‐N‐glycosidase F (PNGase F;Boehringer Mannheim) in phosphate buffer [0.2M Na2HPO4‐NaOH (pH 7.5) containing 10 mM EDTA, 0.5% NonidetP‐40, and protease inhibitors] for 24 hr at 37°C (SDS concentration during the PNGase F incubation: 0.05%). O‐Glycan chains were analyzed via digestion with α‐2,3,6,8,9‐neuraminidase (sialidase; Calbiochem) and endo‐α‐N‐acetylgalactosaminidase (O‐glycosidase; Calbiochem), according to the manufacturer's instructions. Removal of sugar chains was analyzed by using SDS‐PAGE and immunoblotting as described above.
To determine the amount of N‐linked glycosylation, the cell lysate from rat type 1 MG was first denatured in 1% sodium dodecyl sulfate (SDS) (5 min, 60°C). Deglycosylation was then performed with 60 U ml−1 peptide‐N‐glycosidase F (PNGase F;Boehringer Mannheim) in phosphate buffer [0.2M Na2HPO4‐NaOH (pH 7.5) containing 10 mM EDTA, 0.5% NonidetP‐40, and protease inhibitors] for 24 hr at 37°C (SDS concentration during the PNGase F incubation: 0.05%). O‐Glycan chains were analyzed via digestion with α‐2,3,6,8,9‐neuraminidase (sialidase; Calbiochem) and endo‐α‐N‐acetylgalactosaminidase (O‐glycosidase; Calbiochem), according to the manufacturer's instructions. Removal of sugar chains was analyzed by using SDS‐PAGE and immunoblotting as described above.
7
Quantifying Collagen Types in Callus
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The frozen sections were incubated with mouse monoclonal anti-collagen Type I (diluted 1: 4000, C2456, Sigma-Aldrich, USA), mouse monoclonal anti-collagen Type II (diluted 1: 1000, F57, Daiichi Fine Chemical, Japan), rabbit polyclonal anti-collagen Type X (diluted 1: 1000, LB-0092, LSL, Japan), or mouse monoclonal anti-CD68 (diluted 1: 2000, MCA341GA, Serotec, UK) antibodies. A subsequent reaction was made using the strep-tavidin-biotin-peroxidase complex technique with an Elite ABC kit (diluted 1: 50, PK-610, Vector Laboratories, USA). Then, the 3,30-diaminobenzidine tetrahydrochloride (K3466, Dako Japan, Japan) was used for the visualization of the immunoreaction. The sections were finally counterstained with hematoxylin, washed in water, and coverslipped. They were captured with a light microscope (BX-53, Olympus, Japan) at a magnification of ×4. Type I, II, and X collagen-positive areas were measured as a percentage of total callus area with Adobe Photoshop CS (Adobe Systems Inc., Japan).
8
Immunofluorescence Analysis of Lung Tissue
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Frozen lung tissue sections were fixed in acetone for 20 min, then permeabilized by 0.3% Triton X-100 (Sigma, St. Louis, USA) for 15 min and blocked by 10% goat serum for 30 min. Subsequently, sections were incubated with anti-CD68 antibody (BIO-RAD, MCA341GA 1:100 dilution) overnight at 4°C, anti-CD86 antibody (ABclonal, A11991, 100 dilution), and anti-CD206 antibody respectively (Abcam, ab64693, 1:100 dilution). Sections were washed with PBS followed by incubation with fluorescent secondary antibodies (Abcam, ab150116, ab150077) in dark for 1 h at room temperature. Finally, the sections were re-stained with 4’,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. Fluorescent images were captured under a fluorescent microscope (EVOS FL Auto Cell Imaging System, USA).
9
Immunohistochemical Analysis of Tissue Markers
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Samples were washed with PBS three times and then blocked with 5% BSA solution for 30 min, followed by incubation with the primary antibodies diluted in 5% BSA solution overnight at 4 °C: eNOS (ab5589, Abcam), alpha smooth muscle actin antibody (αSMA, ab7817, Abcam), CD68 (MCA341GA, BioRad), Cyclin D1 (A19038, ABclonal), Aggrecan (13880-1-AP, Proteintech), and CD206 (ab64693, Abcam). After that, samples were washed with PBS three times and incubated with goat anti-rabbit secondary antibody or goat anti-mouse antibody for 1 h at room temperature. Nuclei were counterstained with DAPI. Tissue sections without primary antibody incubation were used as negative controls. The stained samples were observed with an inverted microscope (IX73, Olympus, Japan). Five different tissue sections (n = 5) from five different rats for each group were quantified. Specifically, for CD68+ and CD206+ cell quantification, two different tissue sections from the same sample were stained with CD68 and CD206 antibodies, respectively. The numbers of the CD68+ and CD206+ cells and the ratio of CD206+ cells to CD68+ were then quantified. Five sets of tissues sections (n = 5) from five different rats for each group were quantified.
10
Evaluating Cell Transplantation Effects on Gut Microbiome after SCI
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To evaluate whether cell transplantation ameliorates translocation of the intestinal tract bacteria after SCI, fluorescence in situ hybridization (FISH) for the bacterial mRNA in the submucosal layer of ileum 3 and 7 d after SCI [26 (link)], and liver culture 7 d after SCI were performed [9 (link)]. Microbiota sequencing was further assessed to evaluate the change of gut microbiota by collecting feces and small intestinal contents 3 and 14 d after SCI. 16S rRNA gene expressions were amplified with MiSeq Reagent Kit V3 (Illumina, CA, USA) and evaluated with microbial diversity analysis using the Qiime followed by Simpson’s index.
Inflammation cytokines, IL-6 and IFN-γ, were quantified from blood samples of the animals up to 7 d after SCI using commercial ELISA kits for detecting acute systemic inflammation. Chronic systemic inflammations were evaluated by the change of the thymus size [14 (link)], and by immunohistochemistry of macrophages in the spinal cord (anti-CD68 antibody, 1:500, MCA341GA; Bio-Rad Laboratories, Inc., CA, USA) [27 (link), 28 (link)] at 28 d after SCI
Inflammation cytokines, IL-6 and IFN-γ, were quantified from blood samples of the animals up to 7 d after SCI using commercial ELISA kits for detecting acute systemic inflammation. Chronic systemic inflammations were evaluated by the change of the thymus size [14 (link)], and by immunohistochemistry of macrophages in the spinal cord (anti-CD68 antibody, 1:500, MCA341GA; Bio-Rad Laboratories, Inc., CA, USA) [27 (link), 28 (link)] at 28 d after SCI
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