Mouse anti mct1

Paraffin sections from biopsy specimens from resistant/refractory MM patients (n = 3) and early‐stage MM patients (n = 4) were de‐paraffinized and rehydrated as previously described.³⁴ Sections were permeabilized using 0.3% Triton X and blocked to prevent non‐specific antibody binding using a 0.3% Triton X‐10% NGS solution. The slides were then incubated overnight at 4°C with the primary antibodies mouse anti‐MCT1 (Abcam) and rabbit anti‐CD138 (Abcam) at 1:100 dilution in 0.3% Triton‐X. Subsequently, cells were washed three times in PBS for 5 min and then incubated for 1 h at room temperature with the appropriate combination of fluorescence conjugated secondary antibodies donkey polyclonal anti‐rabbit Alexa Fluor 647 (A32849, Thermo Fisher Scientific; 1:500) and subsequently with goat polyclonal anti‐mouse Alexa Fluor 488 (A21247, Thermo Fisher Scientific 1:500). Samples were then washed in 0.3% Triton X in PBS and nuclei were counterstained with DAPI (1:1000) for 5 min, at room temperature. Slices were mounted with fluorescent mounting medium Permafluor (Thermo Fisher Scientific) and digital images were acquired using a Zeiss Axio Imager Z1 Microscope with Apotome 2 system (Zeiss, Milan, Italy).

Mice were anesthetized with 4% (v/v) isoflurane and perfused transcardially with saline, followed by 4% (w/v) paraformaldehyde in PBS. The brains were removed, postfixed overnight in a solution containing 4% (w/v) paraformaldehyde and 4% (w/v) sucrose in PBS, and cryoprotected in solutions containing 10% (w/v) and 20% (w/v) sucrose in PBS for 1 d each. The brains were then frozen in an embedding compound (Sakura Finetek) on dry ice before coronal sections (20 µm) were cut on a cryostat (CM 1100, Leica). The sections were fixed with 4% (w/v) paraformaldehyde for 30 min, permeabilized with 0.3% (v/v) Triton X-100 for 20 min, and treated with 3% (w/v) bovine serum albumin in PBS for 30 min to block nonspecific binding. Next, the sections were incubated for 3 d at 4°C with the following primary antibodies: rabbit anti-glial fibrillary acidic protein (GFAP; 1:1,000; Millipore), mouse anti-MCT1 (1:250; Abcam), mouse anti-MCT4 (1:50; Santa Cruz Biotechnology), or mouse anti-CD147 (1:50; Santa Cruz Biotechnology). After being washed, the sections were incubated for 1 h at room temperature with the following secondary antibodies: Alexa 488- or Alexa 546-conjugated anti-mouse or -rabbit IgG (Invitrogen). Fluorescence images were obtained using a confocal laser scanning microscope (LSM 780, Carl Zeiss, Jena, Germany).