Cryopreservation freezing solution was obtained from ZENOAQ RESOURCE CO., LTD (Fukushima, Japan). DAPI, Live and dead viability/cytotoxicity kit, and MTT assay kit were purchased from Thermo Fisher Scientific (Rockford, IL, USA). MS Collagen (Type 1 atelo-collagen from porcine skin) was obtained from MSBio, Inc. (Gyeonggi, Korea). Antibodies that recognize MITF (ab20663), Mel A (ab731), H2B (ab52599), S100 (ab4066), and tyrosinase (ab738) were purchased from Abcam (Cambridge, UK). Antibodies specific for actin (sc47778) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). A375 melanoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). A375 cells were cultured in DMEM-high glucose with 10% FBS and 1% PS (penicillin-streptomycin).
Ab738
Manufactured by Abcam
Sourced in United Kingdom
Ab738 is a lab equipment product offered by Abcam. It is a device designed for laboratory use, but the core function of the product is not available in a detailed, unbiased, and factual description.
Automatically generated - may contain errors
Lab products found in correlation
Ab12039, by Abcam (2 mentions)
A0428, by Beyotime (2 mentions)
Bs 12024r, by Bioss Antibodies (2 mentions)
A0521, by Beyotime (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab52599, by Abcam (1 mentions)
Ab20663, by Abcam (1 mentions)
Ab731, by Abcam (1 mentions)
Ab4066, by Abcam (1 mentions)
Mtt assay kit, by Thermo Fisher Scientific (1 mentions)
Live and dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
Ecl plus, by Boster Bio (1 mentions)
A0423, by Beyotime (1 mentions)
4 protocols using ab738
1
Cryopreservation of Melanoma Cell Lines
2
Characterization of Melanogenic Proteins
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Total protein was extracted using protein extraction reagent (BEIBO, cat. no. 3101‐50T, China), and protein concentrations were determined using a NanoDrop 1000 spectrophotometer. Western blots were performed according to the usual protocol. In short, proteins were separated by 10% SDS‐PAGE, and transferred to polyvinylidene difluoride membranes. The membranes were cut prior to hybridization with antibodies and then probed with antibodies against TYR (1:1000, cat. no. ab738, Abcam), TYRP1 (1:1000, cat. no. ab 235447, Abcam), TYRP2 (1:1000, cat. no. ab 221144, Abcam), and MITF (1:1000, cat. no. ab140606, Abcam). The membranes were then briefly washed and incubated with a fluorescent secondary antibody. Finally, the membranes were washed and visualized using super enhanced chemiluminescence (ECL) plus (Boster, Wuhan, China). This experiment was performed three times.
3
Immunofluorescence Analysis of Melanocyte Markers
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Samples were processed after the explants had been irradiated with UVR for 48 h. Samples were immediately placed in 4% paraformaldehyde at 4 °C overnight, washed with PBS and then paraffin embedded. Sections were cut at 5–8 µm, dried at 37 °C, deparaffinized in xylene, hydrated in a graded series of ethanol, and subjected to microwave EDTA antigen retrieval. Samples were blocked with 5% bovine serum albumin and 0·5% Tween‐20 in PBS. Slides were incubated with primary antibodies overnight at 4 °C. Incubation with fluorescence‐labelled secondary antibody was for 50 min at room temperature. Primary and secondary labelled antibodies used were as follows: anti‐TYR mouse monoclonal antibody (ab738; Abcam) conjugated to Cy3‐labelled goat antimouse IgG (A0521; Beyotime); anti‐MITF mouse monoclonal antibody (ab12039; Abcam) conjugated to Cy3‐labelled goat antimouse (A0521; Beyotime); anti‐MelanA mouse monoclonal antibody (ab140503; Abcam) conjugated to Cy3‐labelled goat antimouse (A0521, Beyotime); and anti‐OPN5 rabbit polyclonal antibody (bs‐12024R; Bioss) conjugated to Alexa Fluor 488‐labelled goat antirabbit (A0428; Beyotime). (See Figure S2 i.)
4
Immunohistochemical Analysis of Melanocytic Markers
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Cells were inoculated onto coverslips at a density of 1·2 × 104 cells per well. After the cells were irradiated with UVR, they were washed three times with PBS. The cells were fixed with 95% ethanol at room temperature for 10 min and then dried at room temperature. Cells were blocked with 10% FBS in PBS for 30 min In a 5% CO2 incubator at 37 °C. Primary antibodies were incubated overnight at 4 °C. Incubation with fluorescence‐labelled secondary antibody was for 45 min at room temperature. Primary and secondary labelled antibodies used were as follows: anti‐TYR mouse monoclonal antibody (ab738; Abcam, Cambridge, UK) conjugated to Alexa Fluor 488‐labelled goat antimouse IgG (A0428; Beyotime Biotechnology, Haimen, Jiangsu, China); anti‐MITF mouse monoclonal antibody (ab12039; Abcam) conjugated to Cy3‐labelled goat antimouse IgG (A0521; Beyotime); and anti‐OPN5 rabbit polyclonal antibody (bs‐12024R; Bioss, Beijing, China) conjugated to Alexa Fluor 488‐labelled goat antirabbit IgG (A0423, Beyotime). Nuclear staining was performed with 4′,6‐diamidino‐2‐phenylindole. Cells were mounted and visualized under a confocal microscope (Carl Zeiss AG, Oberkochen, Germany). (See Figure S2 g, h.)
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