Rabbit anti glut 1

After PDT treatment, UM-UC-3 and HT 1376 cells were washed twice with ice-cold PBS and harvested in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, 0.5% sodium deoxycholate (DOC), 0.1% SDS, 2 mM phenylmethanesulfonyl (PMSF), 2 mM iodoacetamide (IAD),) and 1× protease inhibitor cocktail (Roche, Indianapolis, IN, USA)). After centrifugation at 16,000 g for 10 min at 4°C, supernatants were used for protein quantification using the Pierce BCA Protein Assay Kit, followed by denaturation of the sample with Laemmli buffer. For the Western Blotting analysis, 60 µg proteins were loaded per lane on sodium dodecyl sulphate-polyacrilamide gels (SDS-PAGE). Following electrophoresis and transfer to PVDF membranes (Bio-Rad, Hercules, CA, USA), the blots were incubated in 5% (m/v) nonfat milk in TBS-T (20 mM Tris, 150 mM NaCl, Tween 0.2%, pH 7.6) and probed with rabbit anti-galectin-1 1∶1,000 (Abcam, Cambridge, UK), rabbit anti-GLUT1 1∶1,000 (Chemicon, Boston, MA, USA) and mouse anti β-actin 1∶20,000 (Sigma) antibodies. After washing, the membranes were probed with secondary anti-rabbit or anti-mouse IgG-HRP-linked antibodies (1∶10,000; Bio-Rad). Immunoreactive bands were detected by enhanced chemiluminiscence (ECL) substrate using an imaging system (VersaDoc 4000 MP, Bio-Rad) followed by densitometric analysis.

Double immunohistofluorescence was performed to distinguish AEF from endothelial cells in the BBB. Primary antibodies used in this experiment were mouse anti-GFAP (a marker for astrocytes) (1:1000) (Chemicon, Temecula, CA, USA) and rabbit anti-GLUT-1 (a marker for endothelial cells) (Chemicon). As described previously [38 (link)], the sections were immunoreacted with a mixture of Alexa Fluor^® 488-conjugated donkey anti-mouse IgG (1:500) (Invitrogen, Waltham, MA, USA) and Alexa Fluor^® 546-conjugated goat anti-rabbit IgG (1:500) (Invitrogen, Waltham, MA, USA). The immunoreacted sections were mounted onto slide glasses and dehydrated in a dry oven of WiseVen^® WOC High Clean Air Oven (Daihan Scientific Co Ltd., Gangwon, Korea). Finally, the sections were coverslipped with Canada balsam (Kanto Chemical Co Inc, Tokyo, Japan).
The double immunoreaction (GFAP/GLUT-1) was observed using confocal MS (LSM510 META NLO) from Carl Zeiss (Oberkochen, Germany) located in the Korea Basic Science Institute Chuncheon Center (Chuncheon, Kangwon, Korea).

To distinguish astrocyte endfeet from endothelial cells in BBB, double immunofluorescence was conducted, as previously described [8 (link),47 (link)]. In brief, the sections were immunostained with primary antibodies: mouse anti-GFAP (diluted 1:1000; Merck-Millipore, Burlington, MA, USA) and rabbit anti-GLUT-1 (a marker for endothelial cells) (diluted 1:100; Chemicon, Temecula, CA, USA) at 4 °C for nine hours. After briefly washing, the sections were incubated in the mixture of goat anti-rabbit IgG conjugated with IgG Alexa Fluor^® 546 (diluted 1:500; Invitrogen, Waltham, MA, USA) and donkey anti-mouse IgG conjugated with Alexa Fluor^® 488 (diluted 1:500; Invitrogen). Thereafter, the sections were washed and dehydrated. The immunostained sections were finally coverslipped with DPX (Sigma-Aldrich Co., St. Louis, MO, USA).
The double immunoreaction of GFAP and GLUT-1 was observed using a Zeiss LSM510 confocal microscope (Carl Zeiss, Oberkochen, Germany), which was located in the Korea Basic Science Institute (KBSI; Chuncheon, Korea).