The optimal CSPG4 staining was performed as follows: After the slides (containing melanoma cell lines spiked into PBMCs or patient blood samples) were thawed and fixed with 2% neutral buffered formalin solution (VWR, San Dimas, CA, USA), non-specific binding sites were blocked with 10% goat serum (Millipore, Billerica, MA, USA). Slides were subsequently incubated with CSPG4-specific mAbs (5 μg/ml total concentration) and Alexa Fluor® 647 pre-conjugated anti-CD45 antibody (MCA87A647, AbD serotec, Raleigh, NC, USA) for 40 min at 37 °C. Slides were then incubated with Alexa Fluor® 555 goat anti-mouse IgG1 antibody (A21127, invitrogen) for 20 min at 37°C. Cells were counterstained with Hoechst 33258 and mounted with an aqueous mounting media. The CSPG4/HMB-45 staining protocol includes minor modifications. After cells were incubated with Alexa Fluor® 555 goat anti-mouse IgG1 antibody (A21121, invitrogen), they were permeabilized using cold methanol for 5 min at RT. Then, cells were incubated with HMB-45-specific mAb (1 μg/ml) for 40 min at 37 °C and subsequently with Alexa Fluor® 488 goat anti-mouse IgG1 antibody.
Alexa fluor 555 goat anti mouse igg1 antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor® 555 goat anti-mouse IgG1 antibody is a secondary antibody conjugated with the Alexa Fluor® 555 fluorescent dye. It is designed to detect and bind to the IgG1 subclass of mouse immunoglobulins, enabling visualization and detection in various immunoassays and imaging applications.
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2 protocols using alexa fluor 555 goat anti mouse igg1 antibody
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Optimized CSPG4 and HMB-45 Staining Protocol
2
Multiparametric Immunofluorescence Staining
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Slides underwent fluorescent staining as described previously [2 (link),28 (link)]. In short, cells were incubated with an antibody mix consisting of conjugated mouse anti-human CD45 Alexa Fluor® 647 (clone: F10-89-4, MCA87A647, AbD Serotec, Raleigh, NC, USA), a cocktail of mouse IgG1/Ig2a anti-human cytokeratins (CK) 1, 4, 5, 6, 8, 10, 13, 18, and 19 (clones: C-11, PCK-26, CY-90, KS-1A3, M20, A53-B/A2, C2562, Sigma, St. Louis, MO, USA), mouse IgG1 anti-human CK 19 (clone: RCK108, GA61561-2, Dako, Carpinteria, CA, USA), and rabbit IgG anti-human vimentin (Vim) Alexa Fluor® 488 conjugated (clone: D21H3, 9854BC, Cell Signaling, Danvers, MA, USA) for 2 h. Slides were then incubated with Alexa Fluor® 555 goat anti-mouse IgG1 antibody (A21127, Invitrogen, Carlsbad, CA, USA) and counter-stained with 4′,6-diamidino-2-phenylindole (DAPI; D1306, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. Slides were finally mounted with a glycerol-based aqueous mounting media to enable future coverslip removal for downstream genomic and proteomic analyses without disrupting cell integrity.
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