HUVEC cells were grown on sterile, round 12-mm glass coverslips (Hecht-Assistent, Sondheim, Germany) coated with rat-tail collagen (BD Biosciences, San Jose, CA, USA) in 24-cluster wells. After treatment, cells were washed with PBS, pH 7.4 (Sigma Chemical Co.), fixed with 2.5% paraformaldehyde in PBS for 15 min and permeabilized with 0.1% Triton X-100 (Sigma Chemical Co.) in PBS for 5 min at room temperature. After washing, nonspecific binding sites were blocked with 10% goat serum and 1% bovine serum albumin (Sigma Chemical Co.) in PBS for 1 h at room temperature. Adherens junction protein staining was performed with a polyclonal rabbit anti-human β-catenin antibody (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C at a dilution of 1: 50 in PBS with 1% goat serum and 0.1% bovine serum albumin. As a secondary antibody a Cy3-labeled polyclonal sheep anti-rabbit antibody (IgG fraction; Abcam, Cambridge, UK) was used at a dilution of 1:50. Nuclear counterstaining was performed with 10 μg/mL 4,6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co.). After washing, coverslips were mounted with ProLong Gold antifade reagent-mounting medium (Molecular Probes, Invitrogen, Eugene, OR, USA) and visualized using a Zeiss Observer.Z1 with a Colibri.2 and ApoTome.2 system (Carl Zeiss AG, Feldbach, Switzerland) at a 400 times original optical magnification.
Prolong gold antifade reagent mounting medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
ProLong Gold antifade reagent mounting medium is a water-based solution used to mount fluorescently labeled samples for microscopy. It is designed to reduce photobleaching of fluorescent dyes during imaging.
Automatically generated - may contain errors
Lab products found in correlation
Antibody diluent block ard1001, by PerkinElmer (4 mentions)
Bond rx, by Leica camera (3 mentions)
Opal polymer hrp ms rb arh1001, by PerkinElmer (2 mentions)
Goat anti mouse poly hrp secondary antibody, by Thermo Fisher Scientific (2 mentions)
Leica bond er2, by Leica camera (2 mentions)
Goat anti rabbit poly hrp secondary antibody, by Thermo Fisher Scientific (2 mentions)
Leica bond rx, by Leica camera (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Rat tail collagen, by BD (1 mentions)
Bovine serum albumin (bsa), by Cell Signaling Technology (1 mentions)
Colibri 2, by Zeiss (1 mentions)
Rabbit anti human β catenin antibody, by Cell Signaling Technology (1 mentions)
Apotome 2 system, by Zeiss (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Phalloidin ifluor 488, by Abcam (1 mentions)
Sulfo cy5 nhs, by Lumiprobe (1 mentions)
Tcs sp2 confocal microscope, by Leica camera (1 mentions)
Ar9 buffer, by PerkinElmer (1 mentions)
8 protocols using prolong gold antifade reagent mounting medium
1
Visualization of Adherens Junctions in HUVEC Cells
2
Evaluating Filter-Grown Cell Cultures
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To evaluate the tightness of filter-grown cell cultures, cells were labeled at the apical or basolateral surface using 1 mM sulfo-NHS-Cy5 (Lumiprobe) in PBS, pH 8.0 for 15 min on ice in the dark and washed three times with PBS before fixation. For all microscopy samples, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.1% TritonX-100 in blocking buffer (1% FBS, 1% BSA in PBS with 0.02% sodium azide) for 10 min at room temperature. Samples were blocked for 1 h at room temperature or overnight at 4 °C in a blocking buffer. Cells were subsequently incubated with mouse anti-ZO1-AF555 antibody (Thermo Invitrogen MA3-39100-A555, 1:100) for 1 h with shaking. Nuclei were stained with 1 µg/mL Hoechst (Molecular probes H1399), and F-actin was stained with phalloidin-iFluor488 (Abcam, 1:1000) for 15 min at room temperature. Samples were fixed with 4% paraformaldehyde for 10 min at room temperature and mounted with Prolong Gold Antifade reagent mounting medium (Molecular Probes). Images were taken with a Leica TCS SP2 confocal microscope and processed using the FIJI software.
3
Multiplex Immunofluorescence Staining of FFPE Tissues
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At 62°C, 4 µm formalin-fixed paraffin-embedded (FFPE) tissue sections were baked for 3 hours with subsequent deparaffinization performed on the Leica Bond RX followed by six sequential cycles of staining, with each round including a 30 min combined block and primary antibody incubation (Perkin Elmer antibody diluent/block ARD1001). For all antibodies other than CD68, detection was performed using a secondary horseradish peroxidase (HRP)-conjugated polymer (Perkin Elmer Opal Polymer HRP Ms+Rb ARH1001; 10 min incubation). Detection of the CD68 primary antibody was performed using a goat antimouse Poly-HRP Secondary Antibody (Invitrogen B40961; 10 min incubation). PanCK, CK7 and Cam5.2 primary antibodies were used as a cocktail. The HRP-conjugated secondary antibody polymer was detected using fluorescent tyramide signal amplification using Opal dyes 520, 540, 570, 620, 650 and 690 (Perkin Elmer FP1487a, FP1494a, FP1488a, FP1496a, FP1495a, FP1497a). The covalent tyramide reaction was followed by heat-induced stripping of the primary/secondary antibody complex using Perkin Elmer AR9 buffer (AR900250ML) at 100°C for 20 min preceding the next cycle. After six sequential rounds of staining, sections were stained with Hoechst (Invitrogen 33342) to visualize the nuclei and mounted with ProLong Gold antifade reagent mounting medium (Invitrogen P36930).
4
Multiplex Immunohistochemistry Staining Protocol
5
Multiplex IF Profiling of TME
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We performed three multiplex immunofluorescence assays to characterize the TME before and after treatment in order to assess T cell exhaustion (CD8, FOXP3, Ki-67, PD-1, LAG3), regulatory T cells (CD3, CD8, FOXP3, GITR, ICOS) and macrophages (CD68, CD3, C3aR, PD-L1, IDO). All panels included a tumor marker (AE1/AE3). 4 um sections obtained from tissue blocks were baked for 2 h at 62 degrees with deparaffinization performed on the Leica Bond RX followed by six sequential rounds of staining, each round including a combined block with primary antibody (PerkinElmer antibody diluent/block ARD1001) followed by a corresponding secondary horseradish peroxidase (HRP)-conjugated polymer (PerkinElmer Opal polymer HRP Ms + Rb ARH1001). Each HRP-conjugated polymer induces the covalent binding of fluorophores using tyramide signal amplification. The covalent reaction was followed by heat induced stripping of the primary antibody in citric acid buffer (pH 6.0; Leica ER1) for 20 min at 100 degrees before the next step in the sequence. For each of the three staining panels, the six antibodies were sequentially stained and then were stained with Spectral DAPI (Perkin Elmer) to visualize nuclei and mounted with ProLong Gold antifade reagent mounting medium (Invitrogen).
6
Multiplex Immunofluorescence Staining Protocol
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FFPE tissue sections were baked for 3 hours at 62 °C in a vertical slide orientation with subsequent deparaffinization performed on the Leica Bond RX, followed by 30 minutes of antigen retrieval with Leica Bond ER2, followed by six sequential cycles of staining with each round including a 30-minute combined block and primary antibody incubation (Akoya antibody diluent/block). For Ki-67 and panCK, detection was performed using a secondary horseradish peroxidase (HRP)-conjugated polymer (Akoya Opal polymer HRP Ms+Rb; 10-minute incubation). Detection of all other primary antibodies was performed using a goat anti-mouse Poly HRP secondary antibody or goat anti-rabbit Poly HRP secondary antibody (Invitrogen, 10-minute incubation). The HRP-conjugated secondary antibody polymer was detected using fluorescent tyramide signal amplification using Opal dyes 520, 540, 570, 620, 650 and 690 (Akoya Biosciences). The covalent tyramide reaction was followed by heat-induced stripping of the primary/secondary antibody complex using Akoya AR9 buffer and Leica Bond ER2 (90% AR9 and 10% ER2) at 100 °C for 20 minutes preceding the next cycle. After six sequential rounds of staining, sections were stained with Hoechst 33342 (Invitrogen) to visualize nuclei and mounted with ProLong Gold antifade reagent mounting medium (Invitrogen).
7
Fluorescent Cell Labeling and Imaging
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As previously described [9 (link)], living cells were labelled with at 5 μg/ml Alexa 488-conjugated WGA (Invitrogen) for 10 min at 37 °C in the CO2 incubator. For structural studies, cells were fixed in 4% paraformaldehyde for 5 min and observed in ProLong Gold antifade reagent mounting medium (Invitrogen).
8
Multiplex Immunofluorescence Staining Protocol
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