To generate brain metastasis, mice were inoculated intracardially as previously described [6 (link)]. Six weeks following the inoculation, mice were perfused with 4% paraformaldehyde, sacrificed, and brains were fixed and embedded in optimal cutting temperature compound (Tissue-Tek® O.C.T., Sakura Finetek USA, Inc., Torrance, CA, USA).
OCT-embedded 10 µm brain sections were cut. The sections were blocked for 30 min. with CAS-BlockTM solution (008120, Life Technologies, Carlsbad, CA, USA). Primary antibodies (Abs) were incubated in Ab diluent (ab64211, Abcam, Cambridge, UK) with 0.02% Triton X-100 overnight at 4 °C, followed by fluorescently conjugated secondary Abs in 0.02% Triton X-100 in PBS for 60 min at RT. Coverslips were mounted using 4′,6-diamidino-2-phenylindole (DAPI) Fluoromount (Southern Biotech, Birmingham, AL, USA). The images were acquired with a × 40/1.10 water objective, using a Leica SP8 confocal microscope and Leica SP8 software (LAS-X v3.5.7.23225, Leica Microsystems, Wetzlar, Germany).
Information on primary Abs against JunB, Iba-1, and melanoma markers is detailed inTable S1 .
OCT-embedded 10 µm brain sections were cut. The sections were blocked for 30 min. with CAS-BlockTM solution (008120, Life Technologies, Carlsbad, CA, USA). Primary antibodies (Abs) were incubated in Ab diluent (ab64211, Abcam, Cambridge, UK) with 0.02% Triton X-100 overnight at 4 °C, followed by fluorescently conjugated secondary Abs in 0.02% Triton X-100 in PBS for 60 min at RT. Coverslips were mounted using 4′,6-diamidino-2-phenylindole (DAPI) Fluoromount (Southern Biotech, Birmingham, AL, USA). The images were acquired with a × 40/1.10 water objective, using a Leica SP8 confocal microscope and Leica SP8 software (LAS-X v3.5.7.23225, Leica Microsystems, Wetzlar, Germany).
Information on primary Abs against JunB, Iba-1, and melanoma markers is detailed in
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