Indirect immunofluorescence was performed as previously described44 (link). Cells grown on glass coverslips for 1 and 2 wks and porous membranes at the A–L and L–L interface for 1, 2 and 4 wks in DMEM, A-DMEM and UroM media were fixed at room temperature with cold (− 20 °C) 100% ethanol for 25 min. After fixation, cells were washed with PBS and incubated in blocking solution containing 1% Bovine Serum Albumin (BSA, Sigma) in PBS for 45 min at room temperature. Primary and secondary antibodies were diluted in 1% BSA in PBS and incubated overnight at 4 °C and 1.5 h at room temperature, respectively. Extensive washing with PBS was performed between each step. Finally, cells were mounted in DAPI-Vectashield medium (Vector Laboratories, Burlingame, CA, USA). For negative controls, incubation with primary antibodies was omitted.
Dapi vectashield medium
Manufactured by Vector Laboratories
Sourced in United States
DAPI-Vectashield medium is a mounting medium developed by Vector Laboratories. The core function of this product is to provide a solution for mounting and preserving fluorescently labeled biological samples for microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Neg 50 frozen section medium, by Thermo Fisher Scientific (1 mentions)
Eclipse ni e microscope, by Nikon (1 mentions)
Guinea pig anti insulin, by Abcam (1 mentions)
Cell m imaging software, by Olympus (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Dm2500, by Leica (1 mentions)
Tissue tek oct, by Sakura Finetek (1 mentions)
Alexa fluor 488 actin conjugate, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
6 protocols using dapi vectashield medium
1
Indirect Immunofluorescence Staining Protocol
2
Immunofluorescence Staining of Pancreatic Tissue
3
Immunofluorescence Imaging of PAD Enzymes
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PC3 and PNT2 cells (5×105 cells/well) were seeded on a cover slip in a 24-well plate in triplicate, incubated for 24 h, washed gently with prewarmed PBS, fixed with 4% PFA for 10 min at RT, washed 3 times with cold PBS and resuspended in PB for 5 min at RT. The buffer was then removed and the cells were washed 3 times as before.
After incubation with PAD2 or PAD4 primary antibodies (1:500 dilution in 3% BSA/PBS) for 1 h at 4°C on a shaking platform, the cells were washed 3 times with cold PBS and further incubated with AlexaFluor 488 conjugated anti-rabbit IgG secondary antibody (Invitrogen; 5 µg/ml in 3% BSA/PBS) at 4°C for 1 h on a shaking platform in the dark. The cells were then washed 3 times with cold 1% BSA/PBS and the cover slips mounted on to slides with DAPI-VECTASHIELD medium (Vector Laboratories, Inc., Burlingame, CA, USA). Images were acquired using a fluorescence microscope (1X81 motorized inverted fluorescence microscope, Olympus Corporation, Hamburg, Germany). The mean green fluorescence intensity of the fluorescence images was analysed as per the manufacturer's instructions using the Cell∧M imaging software (Olympus Corporation) provided with the Olympus 1X81 fluorescence microscope (Olympus Corporation).
After incubation with PAD2 or PAD4 primary antibodies (1:500 dilution in 3% BSA/PBS) for 1 h at 4°C on a shaking platform, the cells were washed 3 times with cold PBS and further incubated with AlexaFluor 488 conjugated anti-rabbit IgG secondary antibody (Invitrogen; 5 µg/ml in 3% BSA/PBS) at 4°C for 1 h on a shaking platform in the dark. The cells were then washed 3 times with cold 1% BSA/PBS and the cover slips mounted on to slides with DAPI-VECTASHIELD medium (Vector Laboratories, Inc., Burlingame, CA, USA). Images were acquired using a fluorescence microscope (1X81 motorized inverted fluorescence microscope, Olympus Corporation, Hamburg, Germany). The mean green fluorescence intensity of the fluorescence images was analysed as per the manufacturer's instructions using the Cell∧M imaging software (Olympus Corporation) provided with the Olympus 1X81 fluorescence microscope (Olympus Corporation).
4
Quantifying Lipid Accumulation in HepG2 Cells
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To assess intracellular lipid accumulation, HepG2 cells seeded on coverslips were exposed to stimuli for 4 h, fixed by using 4% PFA for 10 min, and stained with Nile Red for 15 min. After washing three times with PBS, 5 min each, specimens were counterstained and mounted in DAPI Vectashield medium (Vectorlab, H1500, Newark, CA, USA). Images were captured using a Zeiss LSM710 spectral confocal microscope (Carl Zeiss, Gottingen, Germany). Excitation at 543 nm (HeNe laser) and 405 nm (argon ion laser), and emissions were detected and analyzed. Correlated Total Cell Fluorescence (CTCF) was calculated as follows:
5
Quantitative Confocal Analysis of Occludin in Brain Tissue
6
Liver Ischemia-Reperfusion Injury Assessment
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