The skin explants were fixed with neutral-buffered 10% formalin overnight, washed with PBS, and then embedded in paraffin. Sections were cut into 3-µm thick slices, deparaffinized in xylene, hydrated in a graded series of ethanol, and subjected to antigen retrieval by the microwave method with EDTA. The samples were blocked with 10% bovine serum albumin and 0.5% Tween-20 in PBS. The slides were incubated overnight with primary antibodies at 4 °C. Incubation with fluorescence-labeled secondary antibodies was performed for 30 min at 37 °C. The following primary and secondary labeled antibodies were used: rabbit polyclonal to OPN3 N-terminal (ab228748; Abcam) conjugated to Alexa Fluor 488-labeled goat anti-rabbit IgG (A0423; Beyotime); mouse anti-human pan-cytokeratin monoclonal antibody (sc-81703; Santa Cruz Biotechnology, Inc.) conjugated to Cy3-labeled goat anti-mouse IgG (A0521; Beyotime); rabbit anti-human Dync1i1 polyclonal antibody (AB_2846296, Affinity Biosciences, Ltd.) conjugated to an Alexa Fluor 488-labeled goat anti-rabbit IgG (A0423; Beyotime); and rabbit anti-human DCTN1 polyclonal antibody (AB_2838577, Affinity Biosciences, Ltd.) conjugated to Alexa Fluor 488-labeled goat anti-rabbit IgG (A0423; Beyotime). Nuclear staining was performed with DAPI. Fluorescent images were collected by fluorescence microscopy (Zeiss, Oberkochen, Germany).
A0423
Manufactured by Beyotime
Sourced in China
The A0423 is a laboratory equipment designed for general scientific applications. It features essential functions to support various experiments and analyses conducted in a research or testing environment.
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Lab products found in correlation
A0460, by Beyotime (4 mentions)
A0428, by Beyotime (4 mentions)
A0521, by Beyotime (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions)
Alexa fluor 488 labeled goat anti rabbit igg, by Beyotime (2 mentions)
C1002, by Beyotime (2 mentions)
Alexa fluor 488 labeled goat anti rabbit igg h l, by Beyotime (2 mentions)
Fluorescence microscopy, by Zeiss (1 mentions)
Sc 81703, by Santa Cruz Biotechnology (1 mentions)
Ab228748, by Abcam (1 mentions)
Nb100 40844, by Novus Biologicals (1 mentions)
Af519 1, by Beyotime (1 mentions)
Ab172620, by Abcam (1 mentions)
Ab179503, by Abcam (1 mentions)
At819, by Beyotime (1 mentions)
Eclipse c1 fluorescence microscope, by Nikon (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Image pro plus 5, by Media Cybernetics (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Ab51520, by Abcam (1 mentions)
24 protocols using a0423
1
Immunofluorescence Staining of Skin Explants
2
KIFC1 Antibody Validation Protocol
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The following antibodies and reagents were used in this study. For immunofluorescence, rabbit monoclonal anti-KIFC1 (1:200, ab172620, Abcam), used in Figures 1 , 2 , 3 , 4 , 5 and Supplementary Figures 1–5 ; rabbit polyclonal anti-KIFC1 (1:200, NB100-40844, Novus Biologicals), used for immunofluorescence only in Supplementary Figure 1G and Figure 6A, 6B ; mouse monoclonal anti-GM130 (1:200, 610823, BD Biosciences); rabbit monoclonal anti-γ-tubulin (1:500, ab179503, Abcam); mouse monoclonal anti-α-tubulin (1:500, AT819, Beyotime). Secondary Alexa Fluor 488-conjugated anti-rabbit antibody (1:500, A0423) and Alexa Fluor 555-conjugated anti-mouse antibody (1:500, A0460, Beyotime. DAPI (Beyotime) was used to visualize the nucleus. For western blot, rabbit monoclonal anti-KIFC1 (1:5000, ab172620, Abcam), rabbit polyclonal anti-KIFC1 (1:200, NB100-40844, Novus Biologicals), mouse monoclonal anti-GAPDH (1:2000, D190090, BBI), rabbit polyclonal anti-β-Actin (1:2000, D110001, BBI), mouse monoclonal anti-Flag antibody (1:2000, AF519-1, Beyotime). Secondary goat-anti-rabbit HRP-conjugated antibody (1:2000, A02028, Beyotime), secondary goat-anti-mouse HRP-conjugated antibody (1:2000, D110087, BBI).
3
Immunofluorescence Analysis of LC3 in H9c2 Cells
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H9c2 cardiomyoblasts were seeded in six-well plates with glass coverslips. After incubation and treatment as described above, the cells were fixed in 4% paraformaldehyde for 10 min and blocked with 5% bovine serum albumin in PBS for 30 min at room temperature. Subsequently, the cells were incubated overnight at 4°C with a specific primary antibody against LC3 (Proteintech #14600-1-AP, China, 1:200), followed by incubation with a secondary antibody (Beyotime #A0423, China, 1:500) at room temperature for 1 h. Nuclei were stained with DAPI for 10 min. Finally, fluorescence images were captured using an Eclipse C1 fluorescence microscope (Nikon, Japan) at corresponding excitation wavelengths, and the images were processed with ImageJ software (NIH, United States).
4
Immunofluorescence Staining of EECs
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The EECs were fixed in 4% paraformaldehyde (Beyotime Institute of Biotechnology) at room temperature for 10 min. After two PBS washes, the cells were permeabilized with 0.1% Triton 100-X (Beyotime Institute of Biotechnology) at room temperature for 30 min. The EECs were then washed with PBS three times and blocked with blocking buffer (P0260, Beyotime Institute of Biotechnology) at 37°C for 30 min. Samples were then incubated with the primary antibodies diluted in blocking buffer overnight at 4°C, followed by incubation for secondary antibodies for 1 h. The primary antibodies used included anti-MAP1LC3B (ab51520, Abcam), anti-NPR3 (ab97389, Abcam), anti-CDH11 (H00001009, Novus), anti-PLXND1 (PA5-47012, Invitrogen), and anti-ORAI1 (ab244352, Abcam). The secondary antibodies used were FITC-labeled goat anti-rabbit IgG (H + L) (A0423, Beyotime Institute of Biotechnology) and Cy3-labeled goat anti-mouse IgG (H + L) (A0521, Beyotime Institute of Biotechnology). The cells were then digitalized on a Leica SP5 confocal microscope (Leica Microsystems, Germany) and analyzed using Image-Pro Plus 5.0 (Media Cybernetics).
5
Chondrocyte Extracellular Matrix Analysis
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Chondrocytes were seeded on glass coverslips overnight, fixed with 4% paraformaldehyde, premeabilized with 0.5% Triton‐X, and then blocked with 1% BSA. The cells were incubated with Col II (1:200, NB600‐844, Novus, MO), Aggrecan (1:100, NB600‐504, Novus, MO), and MMP13 (1:100, NBP1‐45723, Novus, MO) antibodies at 4°C overnight. Chondrocytes were then incubated with goat anti‐rabbit IgG Alexa Fluor 488 (A0423, Beyotime Biotechnology, Shanghai, China) or cy5 dye (P0183, Beyotime Biotechnology, Shanghai, China). Antifade mounting medium with DAPI (P0131, Beyotime, China) was used to stain nuclei.
6
Lung Immunohistochemistry for Inflammation
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Lung sections from the aforementioned groups of rats were washed three times with phosphate-buffered saline and then treated with primary antibodies against F4/80, NLRP3, ASC, caspase 1 and SIRT1 at 4° C overnight. Subsequently, the tissues were probed with a secondary antibody (A0423, Beyotime, 1:500), washed with phosphate-buffered saline and sealed with glycerin (95%). A fluorescence microscope was used to visualize the fluorescence indicators.
7
Comprehensive Histological Analysis of Mouse Testes and Epididymis
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The testes and epididymis of the mouse were collected and weighed, fixed in 4% PFA (paraformaldehyde) solution, and then embedded in paraffin wax to cut into 5-μm-thick tissue slices using a rotary microtome. Three slices of each testis and epididymis sample were stained with H&E (hematoxylin-eosin) for histological analysis. For immunofluorescence (IF) staining, cross sections were deparaffinized with xylene (10 min) and rehydrated in a descending ethanol series (100, 90, 80, and 70% ethanol, 5 min each). Antigen retrieval was performed with 1X Tris-EDTA buffer for 10 min by keeping them in boiling, and then pretreated after being cooled to room temperature. Sections were blocked with 5% BSA for 30 min, and incubated with properly diluted primary antibodies (Supplementary Table S2 ) overnight at 4 °C. Then, the sections were rinsed with PBS and incubated with Alexa Fluor 488 or 555-conjugated secondary antibodies (Beyotime, A0423 or A0460, 1:200) for 2 h at room temperature and DAPI Staining Solution (Beyotime) was used to visualize the nucleus. The images were visualized using a Nikon 80i microscope with NIS-Elements software (Nikon, Tokyo, Japan).
8
Immunofluorescent Staining of Liver Tissues
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Immunofluorescent staining was performed on frozen sections (4 μm thick) fixed with the mixture of methanol and acetone. The sections were blocked with 5% goat serum, and incubated with special primary antibodies (Collagen I, 1:1000, 14695-1-AP, Proteintech; α-SMA, 1:1000, cs19245, Cell Signaling Technology (CST); HIF-1α, 1:1000, cs36169, CST; and CAT, 1:2000, 66765-1-Ig, Proteintech) at 4 °C overnight, followed with Alexa Fluor 488-labeled goat anti-rabbit IgG (H + L) secondary antibodies (1:200, A0423, Beyotime). Finally, slides were mounted with DAPI. All sections were observed and analyzed with a fluorescence microscope (ZEISS, AXIO SCOPE.A1). Paraffin-embedded human liver tissues were rehydrated and then stained using a Sirius Red staining Kit (RS1220, G-CLONE) to monitor collagen distribution.
9
Quantification of CD41 in Lung Tissue
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For measurement of CD41 using immunofluorescence staining was performed. Briefly, the frozen sections of lung tissue samples were washed with PBS twice and then were blocked with 1% bovine serum albumin at room temperature for 30 min. Then the cells and heart tissue samples were incubated overnight with rabbit anti-CD41 primary antibody (ab63983, 1:50; Abcam, Cambridge, UK). After being washed with PBS for three times, the samples were performed with goat anti-rabbit secondary antibody conjugated to Alexa Fluor 647 (A0468, 1:400) and goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (A0423, 1:400) (both from Beyotime Institute of Biotechnology) for 1 h under dark conditions. Then, the cells were counterstained with DAPI (Beyotime Institute of Biotechnology) for 15 min, and the tissue sections were analyzed using an immunofluorescence microscope. Three slides per experimental condition are repeated three times.
10
Immunofluorescence Analysis of Podocytes
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The podocytes were plated in a 24-well plate. When 40% confluent, the cells were fixed, infiltrated and blocked. Each slide was incubated with anti-nephrin (ab216341, 1:500, Abcam, USA) and anti-podocin (ab50339, 1:500, Abcam, USA) at 4 °C overnight. Then the slides were washed three times with PBS and incubated with Alexa Fluor 488 labeled goat anti-rabbit IgG (A0423, 1:100, Beyotime, Shanghai, China) for 45 min at room temperature. Finally, cells were observed under a confocal laser scanning microscope (Olympus, FV 1000, Center Valley, PA, USA).
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