The immunofluorescence staining of brain paraffin sections were performed as previously described [25 (link)]. After blocking with 5% bovine serum albumin, the sections were incubated with anti-MRTF-A (1:100, sc-398675, Santa Cruz, CA, USA), rabbit anti-β-amyloid (Cell Signaling Technology, 1:2000) or rabbit anti-LC3B (1: 500, ab48394, Abcam, MA, USA), respectively. Then, the sections were washed with 0.1M PBS and then incubated with the fluorescence-labeled secondary antibodies (goat anti-mouse/rabbit 1:200, ab150113, ab150077, Abcam, MA, USA) for 30 minutes at 37° C, and then washed with PBS. Next, the nuclei were stained with DAPI (5 μg/ml) for 2 minutes, and then analyzed with a laser scanning confocal microscope (CX31-32RFL, Olympus).
Sc 398675
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-398675 is a laboratory equipment product from Santa Cruz Biotechnology. It is designed for general laboratory use. The core function of this product is to support standard research activities in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Ab48394, by Abcam (1 mentions)
Ab150113, by Abcam (1 mentions)
Rabbit anti β amyloid, by Cell Signaling Technology (1 mentions)
Cx31 32rfl, by Olympus (1 mentions)
Ab150077, by Abcam (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
Normal goat serum (ngs), by Wuhan Boster Biological Technology (1 mentions)
Sp 61, by Santa Cruz Biotechnology (1 mentions)
Immunofluorescence microscope, by Leica (1 mentions)
Sc 33766, by Santa Cruz Biotechnology (1 mentions)
Mrtf a, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 or 568 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab5694, by Abcam (1 mentions)
4 protocols using sc 398675
1
Immunofluorescence Staining of Brain Sections
2
Immunofluorescence Staining of MRTF-A
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Following transfection, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature and then blocked with normal goat serum (Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 20 min at room temperature. Following incubation with the primary antibody (cat. no. sc-398675, mouse anti-MRTF-A; 1:200; Santa Cruz Biotechnology, Inc.) in a humidified chamber overnight at 4°C, cells were incubated with the secondary antibody [cat. no. BA1101; fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG; 1:100; Wuhan Boster Biological Technology, Ltd.] for 30 min at 37°C. Subsequently, cells were incubated with DAPI (5 µg/ml; cat. no. C1005; Beyotime Institute of Biotechnology) for 15 min at room temperature. Following washing with PBS, the samples were observed under laser scanning confocal microscope (magnification, ×200; Olympus Corporation, Tokyo, Japan).
3
Immunocytochemical Analysis of Osteoclast Proteins
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For immunocytochemical staining, the fixed mature osteoclasts were incubated with primary antibodies against Arp2 (1:50, D221703, BBI, Sangon Biotech, Shanghai, China), GMFB (1:50, SP-61, Santa Cruz, CA, USA), DBP (1:150, D163666, BBI), DNase I (1:100, D222246, BBI), or MKL1 (1:50, sc-398675, Santa Cruz) at 4 °C overnight, followed by Alexa 647- and Alexa 488-conjugated secondary Abs (1:1000, abcam, Cambridge, UK) for 1 h. Images were taken on a Leica TCS SP5 confocal scanning laser microscope (Leica Laser Technik, Solms, Germany).
4
Immunofluorescence Staining of VSMCs
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VSMCs were seeded on glass coverslips in 24-well cell culture plates at a density of 103 cells/cm2, and then incubated overnight in normal cell growth conditions. For immunofluorescence staining, cells were washed with PBS for 3 times followed by fixation and permeabilization in 75% acetone in ethanol for 5 min. Cells were then blocked with 10% goat serum for 1 h and incubated overnight with primary antibodies against SMYD2 (# 9734, Cell Signaling, RRID: AB 10889559), SM α-actin (ab5694, Abcam, RRID: AB 2223021), myocardin (sc-33766, Santa Cruz, RRID: AB 2148748), myocardin-related transcription factor-A (MRTF-A: sc-398675, Santa Cruz, RRID: AB 2142498), and MRTF-B (PA5-37105, Thermo Fisher Scientific, RRID: AB 2553910). Cells were then rinsed with PBS for 3 times and incubated with Alexa Fluor 488 or 568-conjugated secondary antibodies (1:400 dilution, Thermo Fisher). Finally, cells were counter-stained with DAPI (1:10,000 dilution, Sigma) and mounted with mounting medium. Fluorescence images of stained cells were captured via using a Leica immunofluorescence microscope.
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