Immunofluorescence was performed according to standard protocols and as previously described (Zakariyah et al. 2012 (link)). Briefly, fibroblasts were seeded in eight-well chamber slides (Thermo Fisher Scientific; catalog # 154534) for 48 h in DMEM media, rinsed with PBS, fixed with 3% paraformaldehyde (PFA) for 10 min, and permeabilized in 0.5% Triton X-100 for 3 min. Cells were blocked with 5% goat serum and incubated with first primary and then secondary antibodies (diluted in PBS). VECTASHIELD Hard Set Mounting Medium containing DAPI (4′,6-diamidino-2-phenylindole) (Vector; catalog # H-1500) was used to counterstain nuclei. The following primary antibodies were used for immunofluorescence: Anti-PDI [RL90] (1:100; Abcam; catalog # ab2792-100), Anti-TGN38 [21-G (1:250; Santa Cruz Biotechnology; catalog # sc-101273), and LF-68 anti-Pro-COL1A1 rabbit serum (1:200, a gift from L.W. Fisher, National Institutes of Health [NIH]) (Fisher et al. 1995 (link)). Goat anti-mouse IgG 488 (Alexa-Fluor, Invitrogen) and goat anti-rabbit IgG 568 (Alexa-Fluor, Invitrogen) were used as secondary antibodies. Images were recorded with an Olympus 1X81 confocal microscope. The overlap of two proteins on each section was analyzed by Mander's coefficient for green (M1) and red (M2) separately. Colocalization of pro-COL1A1 and PDI or pro-COL1A1 and TGN38 was analyzed from two independent experiments.
Vectashield hard set mounting medium containing dapi
Manufactured by Vector Laboratories
Sourced in United States
VECTASHIELD Hard Set Mounting Medium containing DAPI is a high-performance, anti-fade mounting medium designed for the preservation and visualization of fluorescent-labeled samples. It provides a hard set, long-lasting protective barrier for mounted specimens.
Automatically generated - may contain errors
Lab products found in correlation
Anti pdi rl90, by Abcam (1 mentions)
81 confocal microscope, by Olympus (1 mentions)
Axioplan 2 microscope, by Zeiss (1 mentions)
Axiocam hrm microscope camera, by Zeiss (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Hyaluronidase type 4, by Merck Group (1 mentions)
N nitrosodimethylamine, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
5 protocols using vectashield hard set mounting medium containing dapi
1
Immunofluorescence of Fibroblast Proteins
2
Immunofluorescence Staining of IL-6 Receptor
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LNCaP cells were seeded at 4 × 105 cells and 22Rv1 at 2 × 105 cells per well in 6-well plates with 22 × 22 mm glass slides at the bottom of each well. Cells were allowed to adhere on the glass slides overnight before the immunofluorescence staining was performed. After washing with PBS, cells were fixed with 4% formaldehyde for 10 min, permeabilized with 0.2% Triton for 2 min, and then blocked with 2% BSA in PBS-T for 15 min. The fixed cells were then incubated with the anti-IL-6 receptor primary antibody (1:100) at 4 °C overnight. The next day, cells were washed with PBS and incubated with anti-rabbit Alexa Fluor 594 conjugated secondary antibody (1:1000) (Life Technologies) for an hour at room temperature. Finally, the cells were washed with PBS and mounted with cover slips using Vectashield Hard Set Mounting Medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). Images were captured on a Zeiss AxioPlan 2 microscope connected to an AxioCam HRm microscope camera. Jurkat cells that are known not to express IL-6Rα62 (link) were used as negative control for IL-6 receptor.
3
Isolation of Rat Adipose-Derived Cells
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N-nitroso-dimethylamine, collagenases type I, hyaluronidase type IV, DNase I, penicillin and streptomycin were purchased from Sigma-Aldrich Co. (St. Louis, MO). Growth media and gentamicin were obtained from GIBCO BRL., Life Technologies (Gaithersburg, MD). Collagenase Medium consisted of RPMI 1640 medium containing 0.1% collagenase type I, 0.01% hyaluronidase type IV, 0.01% DNase I, 100 U/ml penicillin and 100 µg/ml streptomicin. Bovine serum albumin (BSA) was the product of Fermentas International Inc. (Burlington, Canada). Polyclonal rabbit anti-rat-GLUT-1, anti-rat-GLUT-3 and anti-TGF ß-1 were bought from Abcam Inc. (Cambridge, MA, USA). Texas red-conjugated anti-rabbit secondary antibody and Vectashield Hard Set mounting medium containing DAPI were from Vector Laboratories, Ltd. (Peterborough, England).
Phospate buffered saline (PBS) contained: 140 mM NaCl, 5 mM KCl, 8 mM Na2HPO4 at pH 7.3. Phosphate buffered saline with Tween (PBST) consisted of 0.1% Tween 20, 20 mM Na2HPO4, 115 mM NaCl; pH 7.4. The collagenase Solution for perfusion contained 30 mg collagenase type IV in 100 ml PBS solution.
Phospate buffered saline (PBS) contained: 140 mM NaCl, 5 mM KCl, 8 mM Na2HPO4 at pH 7.3. Phosphate buffered saline with Tween (PBST) consisted of 0.1% Tween 20, 20 mM Na2HPO4, 115 mM NaCl; pH 7.4. The collagenase Solution for perfusion contained 30 mg collagenase type IV in 100 ml PBS solution.
4
Immunofluorescence Staining of HCV Core
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Medium was aspirated from the wells of the 8-chamber slides and cells were washed by immersing the slide in 1X phosphate-buffered saline (pH = 7.4; PBS) for 2 minutes. The cells were then fixed and permeablized by immersing the slide in 100% acetone for 2 minutes. For HCV core staining, the slides were covered with mouse monoclonal anti-HCV core antibody (B2, Anogen) diluted 1:200 in 5% BSA in PBS for 20 minutes. Slides were washed in PBS for 5 minutes, then incubated for 20 minutes with the secondary antibody (goat anti-mouse Alexa Fluor® 488; Invitrogen) diluted 1:100 in PBS. The slides were then washed and mounted with Vectashield Hard Set mounting medium containing DAPI (Vector Laboratories). The slides were examined at 10X and 20X magnifications on a Zeiss Axio Imager.M2 immunofluorescence microscope.
5
Immunofluorescence Assay for XPO5 and TRP2
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For immunofluorescence assays, cells were seeded in chamber slides, washed with PBS, fixed with pre-chilled acetone for 10 min at −20°C, permeabilized using 0.1% TritonX-100/PBS for 5 min, washed again and blocked for 1 h with 1% BSA/PBS. Subsequently, the cells were incubated with a 1:10 dilution of anti-XPO5 antibody (EX5 4E1) or anti-TRP2 (Sigma) in 1% BSA/PBS overnight at 4°C. After they were washed with PBS three times, the cells were incubated with a 1:50 dilution of FITC-conjugated anti-rat immunoglobulin for 1 h, washed again with PBS and mounted with VECTASHIELD Hard Set Mounting Medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). Images were taken using immunofluorescence microscopy with an Axio Imager Zeiss Z1 fluorescence microscope (AxioVision Rel. 4.6.3, Carl Zeiss AG, Oberkochen, Germany).
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