Alexa fluor 633 goat anti rabbit

Images were acquired using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany) with a 40× objective/1.4 numerical aperture oil PlanApochromat immersion lens. Alexa-fluor 488 fluorescence was detected using the 488 nm laser line of an Ar laser and an LP 505 filter. TRITC fluorescence was detected using a 561 nm HeNe laser (1 mW) and an LP 560 filter and Alexa-fluor 633 fluorescence was detected using a 633 nm HeNe. Z-stacks were acquired to confirm the intracellular localization of TRITC labelled AuNRs. The thickness of the slices and the interval between slices were set to 0.7 µm. After irradiation, cells were deposited onto a glass slide using cytospin centrifugation (800 rpm, 5 min), fixed with 4% PFA for 20 min and stained for confocal microscopy. Cell membrane was stained with monoclonal mouse anti-human CD45 antibody, diluted 1:50 (R&D Systems, MAB1430) and using Alexa Fluor-488 goat anti-mouse igG as secondary antibody, diluted 1:1000 (Life technologies, A11001). For staining early endosomes, cells were permeabilized with 0.1% TRITON X-100 for 10 min and stained with rabbit anti-EEA1 monoclonal antibody (Cell Signaling Technology, 3288), diluted 1:100. Alexa Fluor-633 goat anti-rabbit (Life Technologies, A21070), diluted 1:1000 was used as secondary antibody. Cell nuclei were stained with DAPI.

For post hoc examination of ChR2 expression and optical fiber-placement confirmation, mice were deeply anesthetized by intraperitoneal injection of ketamine/xylazine (100 and 10 mg/kg, respectively) and then transcardially perfused with cold tris-buffered saline (TBS) followed by 4% paraformaldehyde (PFA) in TBS. After overnight post-fixation in PFA, the cerebellum was removed by dissection and cut into thin sections (60–80 μm) that were mounted onto glass slides. To immunohistochemically label PCs, cerebellar slices were incubated for one hour at room temperature in blocking solution (10% normal goat serum and 0.2% Titron X-100 in TBS) and then overnight in primary antibodies (anti-calbindin #CB38a, Swant; 1:1000 in 5% normal goat serum 0.1% Triton X-100 in 1× TBS) at 4 °C. After washing in phosphate-buffered saline, slices were incubated for 1 h at room temperature in secondary antibodies, either Alexa Fluor-633 goat anti-rabbit (1:1000 #A-21070, Thermofisher) or Alexa Fluor-488 goat anti-rabbit (1:1000, #A-27034, Thermofisher), and then mounted on slides after repeat washes in TBS.

HUVECs (5 × 10⁴ cells/well) were plated in glass coverslips, previously coated with fibronectin (1 μg/cm²), in serum-supplemented DMEM and left overnight in an incubator at 37 °C, 5% CO₂. DisBa-01 (1000 nM), previously labelled using Alexa Fluor® 546 dye (Invitrogen, Thermo Scientific), was added to the cells for 2 min. Samples were fixed in 4% paraformaldehyde for 10 min and permeabilized using 0.5% Triton X-100 for 10 min. Samples were washed with PBS, followed by a 1-h incubation in 5% PBS-BSA to block unspecific sites. Cells were incubated overnight with targeted primary antibodies (1:100 Rabbit pAb to VEGF Receptor 2; 1:100 Mouse Monoclonal to the integrin α_vβ₃, Abcam). Then, secondary antibodies (1:1000 Alexa Fluor 633 goat anti-rabbit, ThermoFisher; 1:1000 Goat polyclonal anti-mouse Alexa Fluor 488, ThermoFisher Scientific) were mixed in 5% PBS-BSA and applied on the wells. After incubation, slides were cleaned and samples were stained with DAPI (Thermo Fisher Scientific) for 10 min. Slides were assembled using ProLong™ Antifade Reagents for Fixed Cells (Thermo Fisher Scientific) and observed on confocal microscope Axio Observer LSM 780 (Zeiss) aided by ZEN BLACK software. Analysis occurred under the same laser intensity for different fluorescences at 63x magnification. Colocalization coefficients were determined using ImageJ FIJI program.

Patient samples (LV1221) were purchased from US Biomax (Rockville) and analyzed for the expression of TIPRL, LC3, CD133 and CD46 as the following; briefly, de-paraffinization was performed by immersing each slide in the order of xylene, as well as 100, 95, and 70% ethanol (Merck, 1.00983.1011) for five minutes each. Then the slides were subjected to a pre-heated sodium citrate buffer (0.01 M, pH 6.0; Merck, S4641) for 15 minutes to retrieve antigens. After that, the slides were incubated with antibodies against TIPRL (1:100; Bethyl laboratories, A300-663A), LC3 (1:100; Merck, L7543), CD133 (1:100; NOVUS, NBP2-37741) and CD46 (1:200; SCBT, sc-52647) overnight. The slides were then reacted with Alexa Fluor 633 goat anti-rabbit (1:100, TIPRL; Thermo Fisher Scientific, A11079), Alexa Fluor 568 goat anti-rabbit (1:100, LC3; Thermo Fisher Scientific, A21071) and Alexa Fluor 488 goat anti-mouse (1:100, CD133 and CD46; Thermo Fisher Scientific, T7458), respectively. Confocal observation (ZEISS LSM 800) was followed by quantification of each expression (Supplementary Table 2) using the Carl Zeiss LSM Image program (ZEN 2.3 lite) according to a previous study^{29 (link)}.
Patient information provided by US Biomax (Rockville) was used for this study (Supplementary Tables 1 and 3).

Immunofluorescence was performed using a goat–anti-rabbit antibody conjugated with Alexa Flour 633 (A21070; Thermo Fisher Scientific, Waltham, Massachusetts, USA) for detection of CK7 for the visualization of ACH-3P cells.
Sections with seeded ACH-3P were washed with 1 × PBS and incubated with UV Block (Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 10 min. The primary antibody, polyclonal rabbit CK7, was diluted 1:200 in antibody diluent and incubated for 30 min. Subsequently, slides were washed with 1 × PBS and incubated with the secondary antibody, Alexa Fluor 633 goat–anti-rabbit (1:200; Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 30 min. Finally, the slides were washed, and the nuclei were stained with DAPI (1:2000; Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 5 min. Rabbit immunoglobulin fraction (5 µg/ml, diluted in antibody diluent, Agilent Technologies) served as a negative control. Sections were mounted with ProLong™ Gold Antifade Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA).