All the specimens were deparaffinized and rehydrated. Following antigen retrieval, the tissues were incubated with primary anti-Acox1 (1:100, 10957-1-AP; Proteintech group) and α-SMA (1:200, BM0002; Boster Biotechnology) antibodies at 4 °C overnight. At least ten fields were randomly selected to evaluate the percentage of Acox1-positive and α-SMA-positive RTECs. For immunofluorescence, all sections were incubated with anti-Acox1 antibody (1:100, 10957-1-AP; Proteintech group) followed by Alexa-488 conjugated goat anti-rabbit antibody (1:200, ab150077; Abcam), anti-α-SMA antibody (1:200, BM0002; Boster Biotechnology), and anti-collagen I (1:100, ab6308; Abcam) followed by Alexa-555 conjugated goat anti-mouse antibody (1:200, ab150118; Abcam) at 4 °C overnight. The cells were then co-stained with DAPI (1:500, C1006; Beyotime Biotechnology). All samples were analyzed using a fluorescence microscope (Leica, Germany). At least six fields were randomly selected to evaluate the percentage of positive staining with Image J software (version 1.37; National Institutes of Health, Bethesda, MD).
Bm0002
Manufactured by Boster Bio
Sourced in China, United States
BM0002 is a laboratory centrifuge designed for general purpose applications. It features a compact and durable construction, with a maximum speed of 6,000 RPM and a capacity of up to 4 x 50 mL tubes. The unit is easy to operate and includes a timer function for precise control over the centrifugation process.
Automatically generated - may contain errors
Lab products found in correlation
Ab6308, by Abcam (3 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Ba0325, by Boster Bio (2 mentions)
Ba3414, by Boster Bio (2 mentions)
Biotinylated anti rabbit anti mouse igg, by Boster Bio (2 mentions)
Diaminobenzidine dab, by Boster Bio (2 mentions)
C1006, by Beyotime (2 mentions)
Ab150077, by Abcam (2 mentions)
Ba3638, by Boster Bio (2 mentions)
A00090 1, by Boster Bio (2 mentions)
Ab6328, by Abcam (2 mentions)
Ab108412, by Abcam (2 mentions)
Bm4075, by Boster Bio (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Ab109122, by Abcam (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Ab14106, by Abcam (1 mentions)
Ab15348, by Abcam (1 mentions)
A7811, by Merck Group (1 mentions)
Ab28364, by Abcam (1 mentions)
33 protocols using bm0002
1
Immunohistochemical Analysis of Acox1 and α-SMA in Renal Tubular Epithelial Cells
2
Immunofluorescence Analysis of Renal Cells
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Kidney slides were incubated overnight at 4°C with primary antibodies anti-DppD (1:100, ab187048, Abcam, UK), anti-Claudin-2 (1:100, Product#51-6100, Invitrogen, USA), and anti-α-SMA (1:200, BM0002, BOSTER, China). Dipeptidyl peptidase (DppD) was used as a marker of PTs. Negative controls were incubated with phosphate-buffered saline (PBS). At room temperature, NRK49F cells cultured on coverslips were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% TritonTMX-100 for 10 minutes, and blocked with 2% BSA for 45 minutes. The cells were labeled with antibodies against PCNA (1:200, 10205-1-AP; Proteintech, Wuhan, China), α-SMA (1:200, BM0002, BOSTER, China), and anti-Claudin-2 (1:100, Product#51-6100, Invitrogen, USA). Secondary antibodies Alexa Fluor® 488 donkey anti-rabbit (Product#A32790, Invitrogen, USA), Alexa Fluor® 488 donkey anti-mouse (Product#A-11029, Invitrogen, USA) were used at a 1:300 dilution in vivo and 1:800 dilution in vitro, respectively. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI)(Sigma-Aldrich) according to the manufacturer’s instructions. Images were captured by fluorescence microscopy (Ni-V, Nikon, Japan), and photographs were recorded.
3
Collagen and Fibrosis Immunohistochemistry
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Masson staining was used for collagen (Goodbio Technology Co. Ltd. Wuhan, China). Immunohistochemistry was performed on paraffin-embedded liver sections. Antibodies against SATB1 (Ab109122, Abcam), α-SMA (BM0002, Boster, Wuhan, China) were used for immunohistochemistry. The overall score of each section was assessed by multiplying the intensity score by percentage score of positive staining cells. The classification of each section was determined by the scoring system previously described43 (link).
4
Evaluating Epithelial-Mesenchymal Transition in Renal Tissues
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Immunofluorescence staining of renal tissues was conducted to observe the extent of EMT. Frozen sections were placed in acetone ethanol and fixed for 20 min and then washed with PBS three times. Next, sections were placed in a 3% solution of hydrogen peroxide and incubated in dark for 10 min at room temperature. These were then washed with PBS for three times and dried with 5% bovine serum albumin (BSA) for 20 min. Samples were incubated with mouse anti-α-SMA antibody (BM0002, 1 : 300, Boster) and mouse anti-E-cadherin antibody (GB12082, 1 : 150, Serviceio) at 4°C overnight, and then, goat anti-mouse antibodies were added for 1 h. Subsequently, DAPI was applied for 5 min. Stained sections were imaged at 400x magnification under a fluorescence microscope (MicroPublisher, MP3.3-RTV-CLR-10, Q-IMAGING, Canada). The further quantitative analysis of α-SMA and E-cadherin was performed by IPP.
5
Immunofluorescence Staining of α-SMA in BM-MSCs
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Immunofluorescence staining was conducted with anti-α-SMA (BM0002, Boster Biological Technology, China) as described previously [13 (link)]. α-SMA expression in BM-MSCs was observed under a fluorescence microscope (Zeiss AG, Oberkochen, Germany).
6
Immunofluorescence Staining of Cardiac Cells
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Cells or tissue slices were fixed in 4% paraformaldehyde for 10 minutes, permeabilized with 0.4% (vol/vol) Triton X-100 for 10 minutes, and blocked with 5% bovine serum albumin for 1 hour at room temperature. Then cells or slices were incubated with primary antibody at 4 °C overnight followed by the secondary antibodies for 1 hour at room temperature. The following primary antibodies were used: α-actinin (A7811, Sigma), cTnT (MA512960, ThermoFisher), SM22α (ab14106, Abcam), ACE2 (ab15348, Abcam), CD31 (ab28364, Abcam), and α-SMA (BM0002, Boster). Images were taken using a Zeiss Cell confocal laser scanning microscope 800 (Zeiss).
7
Immunofluorescence Analysis of Autophagy in Aortic Smooth Muscle Cells
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The thoracic aortas were isolated and embedded in optimal cutting temperature compound (Sakura, Japan) for sectioning at an 8-um thickness. Frozen slides were incubated overnight at 4ºC with antibodies against LC3B (NB100-2220; 1:100; Novus Biologicals, CO, USA) and alpha-smooth muscle actin (α-SMA) (BM0002; 1:100; Boster Biological Technology) and then treated with FITC-labeled anti-rabbit IgG (31635; 1:100) and Cy3-labeled anti-mouse IgG (A10521; 1:100) secondary antibodies (Invitrogen, CA, USA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). MASMCs were infected with the mRFP-GFP-LC3 adenovirus for 48 h. The puncta in thoracic aortas and MASMCs were viewed under a confocal microscope (Zeiss LSM800, Carl Zeiss, Munich, Germany) with z-stacks and 63× objective lens. The number of puncta was analyzed using the ImageJ program. For endogenous colocalization studies, MASMCs were incubated with TMEM16A (ab53212; 1:50), VPS34 (ab124905; 1:50) (Abcam), p62 (sc-48402; 1:100), Beclin-1 (sc-48341; 1:1000) (Santa Cruz Biotechnology), and Bcl-2 (BM4985; 1:100) (Boster Biological Technology) antibodies, and processed for colocalization observation with a confocal microscope.
8
Assessing Smooth Muscle Markers Expression
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To assess SM22α and α-SMA expression in cells, IF was employed. Slides were fixed with 4% paraformaldehyde for 30 min, blocked with 0.3% Triton X-100 at 37 ℃ for 30 min, and incubated in a 5% BSA blocking solution for 1 h. They were then incubated overnight at 4 ℃ with primary antibodies against SM22α (ab155272, 1:50, Abcam) and α-SMA (BM0002, 1:50, BOSTER). Subsequently, slides were incubated with secondary antibodies (Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG(H + L) (AWS0005b) and Alexa Fluor 594-conjugated Goat Anti-Mouse IgG(H + L) (AWS0004b)) from Abiowell, diluted 1:200, for 90 min at 37 ℃. Finally, slides were sealed with glycerol and observed using a fluorescence microscope (BA410T, Motic) in a dark environment.
9
Histopathological Liver Tissue Analysis
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For histopathological examination, the liver tissue sections were stained with haematoxylin-eosin (H&E). Sirius red was used to stain for collagen. Immunohistochemistry was performed using 4–5 mm paraffin-embedded thick liver sections, and observed under a photomicroscope. Immunofluorescence staining were observed under a laser scanning confocal fluorescence microscope (Carl Zeiss, Jena, Germany). Antibodies were used as follow: FOXA2 (ab108422, Abcam), α-SMA (BM0002, Boster), α-SMA (MO85129, Dako), CHOP (sc-575, Santa Cruz) and Cleaved Caspase 3 (Asp175,9664, Cell Signaling). Areas of positive stained sites were measured by using image analyses software Image-Pro Plus 6.0 (Media Cybernetics).
10
Quantitative Liver Fibrosis Assessment
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Sirius red was used to stain for collagen. Immunohistochemistry was performed on paraffin-embedded liver sections. Antibodies against HNF1α (ab96777, Abcam), α-SMA (BM0002, Boster, Wuhan, China), SHP-1 (ab2020, Abcam), p-Erk1/2 (Thr202/Tyr204, 4370, Cell Signaling), p-p65 (sc-101752, Santa Cruz) and p-STAT3 (Ser727, 9134, Cell Signaling) were used for immunohistochemistry. Sections were stained with ImmunoCruz™ goat ABC Staining (Sc-2023, Santa Cruz) or EnVision Detection Rabbit/Mouse Kit (GK500710, GeneTech, Shanghai, China) and counterstained with hematoxylin. Areas of positive stained sites were measured using image analyses software Image-Pro Plus 6.0 (Media Cybernetics). Percentage of positive area in corresponding field of liver tissue was calculated to show the intensity of collagen deposition or protein expression.
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