A 5-μm sections of mice frozen tissues were blocked with 10% FBS and incubated with primary antibodies, monoclonal anti-PLAUR/uPAR (1:100, SAB4200412, S igma Aldrich, MO, USA), monoclonal anti-ITGB3 (GPIIIa, CD61), PSI domain [AP-5] (1:100, P05106, Kerafast, USA), and polyclonal anti-synaptopodin (1:200, SC-50459, Santa Cruz, Texas, USA), respectively. The sections were then incubated with an FITC-conjugated anti-mouse secondary antibody (1:200, A0568, Beyotime, Jiangsu, China) or a Cy3-conjugated anti-rabbit antibody (1:200, A0516, Beyotime, Jiangsu, China). The images were captured under the Leica microscope (DM5000B). The results were quantified using 5 different images captured randomly in each group.
Sc 50459
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-50459 is a lab equipment product from Santa Cruz Biotechnology. The core function of this product is to facilitate the handling and processing of biological samples in a laboratory setting. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Dm5000b, by Leica (1 mentions)
A0568, by Beyotime (1 mentions)
A0516, by Beyotime (1 mentions)
Hematoxylin, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Mca341r, by Bio-Rad (1 mentions)
Ab150075, by Abcam (1 mentions)
Ab150105, by Abcam (1 mentions)
Ab6308, by Abcam (1 mentions)
Mab1835, by R&D Systems (1 mentions)
Vectashield mounting medium h 1000, by Vector Laboratories (1 mentions)
Ms 113 p, by Thermo Fisher Scientific (1 mentions)
Sc 271098, by Santa Cruz Biotechnology (1 mentions)
Tcs sp8, by Leica (1 mentions)
4 protocols using sc 50459
1
Immunohistochemical Analysis of Tissue Markers
2
Immunostaining for APLNR and Synaptopodin
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Tissue sections at 5 μm were used to perform immunostaining for APLNR and synapotopodin with rabbit anti APLNR (sc-33823; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-synapotopodin (sc-50459; Santa Cruz Biotechnology). Second antibodies were donkey anti-rabbit-TR and donkey anti rabbit-HRP. Images were obtained using a microscope (Olympus BX-63, Tokyo, Japan).
3
Immunohistochemical Analysis of Kidney Markers
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Paraformaldehyde–fixed, paraffin-embedded sections were used for immunostaining. For single and double staining, paraffin sections were rehydrated and antigen was retrieved by microwave. Sections were incubated with the following primary antibodies at 4 °C overnight: rabbit anti-WT-1 (1:200, RB-9209-P1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-synaptopodin (1:100, sc-50459; Santa Cruz Biotechnology), rabbit anti-CD44 (1:200, 65,294; Progen, Brisbane, Australia), mouse anti-CD68 (1:500, MCA341R; Bio-Rad Laboratories). The tissue was incubated with secondary antibodies for 30 min at RT. Slides were counterstained with hematoxylin or Hoechst 33,258 (Molecular Probes, Darmstadt, Germany) to stain nuclei. WT-1 positive density was expressed as the number of positive cells in each glomerulus divided by the glomerular tuft area. The percentage of double-positive cells (CD44+ and synaptopodin+) per CD44+ cells was calculated, and CD68 positive density was expressed as percentage of area. Positive and negative controls stained appropriately. All tissue assessments were made while unaware of treatment group.
4
Immunostaining Analysis of Kidney Fibrosis Markers
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Kidneys were embedded in OCT (4583, SAKURATissue-Tek®, Torrance, CA, USA) on dry ice. Five-micrometer sections were cut and performed with immunostaining. Briefly, the slices were fixed in 10% neutral buffered formalin; then washed with PBS and treated with 0.2% Triton X-100; mouse anti-αSMA (MS-113-P, Thermofisher, USA) and rabbit anti-synapotodin (sc-50459; Santa Cruz Biotechnology, Santa Cruz, CA, USA); or mouse anti-collagen 1α (ab6308, abcam, USA) and rabbit anti-synapotodin; or mouse anti-fibronectin (sc-271098, Santa Cruz, USA) and rabbit anti-synapotodin; or mouse anti-TGFβ (MAB1835, R&D, USA) and rabbit anti-synapotodin were incubated with the slices after blocked with 1% BSA; Donkey anti-mouse IgG-488 (ab150105; Abcam, Shanghai, China) and donkey anti-rabbit IgG-647 (ab150075; Abcam) were incubated with the slices; Hoechst 33342 was then used to stain the nucleus; the slices were mounted in VECTASHIELD Mounting Medium (H-1000, Vector Laboratories, Inc., Burlingame, CA, USA) staining. Images were obtained using a confocal microscope (TCS-SP8; Leica, Buffalo Grove, IL, USA). Positive cells with both synapotodin and EMT markers located in the glomeruli were counted and divided by synapotodin-positive cells located in the glomeruli. At least 100 glomeruli were analyzed in each group.
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