Cells were seeded on sterile 13‐mm circular glass coverslips placed in 24‐well plates at 20 000 cells per well and incubated for 48 hours. Cells were washed twice with PBS and fixed with 4% paraformaldehyde for 20 minutes. After washed with PBS three times, cells were permeabilized by incubation in PBS containing 0.3% Triton X‐100 for 5 minutes. Cells were washed three times with PBS and incubated in PBS containing 10% goat serum for 1 hour at room temperature. Cells were incubated with the primary rabbit anti‐Piezo1 antibody (Proteintech) at a dilution of 1:100 overnight at 4°C. After washed with PBS three times, cells were incubated with the secondary fluorescein isothiocyanate‐conjugated goat anti‐rabbit IgG antibody at 1:500 at room temperature for 1 hour in dark. After washed with PBS and rinsed with water, cells were mounted on a microscope slide with antifade mounting medium containing 4′,6‐diamidino‐2‐phenylindole (DAPI) (Invitrogen). Fluorescent images were captured using a LMS880 confocal microscope and the ZEN software (Zeiss) or an EVOS Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).
Antifade mounting medium containing 4 6 diamidino 2 phenylindole dapi
Manufactured by Thermo Fisher Scientific
Antifade mounting medium containing 4′,6‐diamidino‐2‐phenylindole (DAPI) is a laboratory product designed to preserve and protect fluorescent samples during microscopy analysis. The core function of this medium is to minimize photobleaching and maintain the integrity of fluorescent signals, particularly those associated with DAPI, a common nuclear stain used in cell and tissue imaging.
Automatically generated - may contain errors
Lab products found in correlation
Anti piezo1 antibody, by Proteintech (1 mentions)
Lms 880 confocal microscope, by Zeiss (1 mentions)
Evos cell imaging system, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Clone cs 1 4, by Agilent Technologies (1 mentions)
2 protocols using antifade mounting medium containing 4 6 diamidino 2 phenylindole dapi
1
Immunostaining of Piezo1 in Cells
2
Immunohistochemistry for EBV Proteins in Multiple Sclerosis
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EBER in situ hybridization was performed using the PNA probe, which hybridizes with both EBER1 and EBER2, and the detection kit from DakoCytomation (Glostrup, Denmark), as described previously (35 (link), 37 (link)).
Immunohistochemical stainings for EBV latent (EBNA2) and lytic (BZLF1) proteins and double immunofluorescence stainings for LMP1 and the B-cell marker CD79a and for BZLF1 and the plasma cell marker IgA, IgG, and IgM were performed in sections from paraformaldehyde (PFA)-fixed frozen brain tissue blocks of three MS donors analyzed in this study (MS79, MS92, and MS121), as previously described (19 (link), 35 (link)– (link)38 (link)). For double immunofluorescence staining for LMP1 and LMP2A, brain sections were incubated with anti-LMP2A rat MAb (1:50; clone TP4E11; Ascenion, Munich, Germany) and anti-LMP1 mouse MAb (1:100; clone CS.1-4; DakoCytomation) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin overnight at 4°C, and, after washing with a mixture of Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen, Eugene, OR) and tetramethylrhodamine (TRITC)-conjugated donkey anti-rat Ig (Jackson ImmunoResearch Laboratories, Cambridgeshire, UK), both diluted 1:300 in PBS containing 3% normal donkey serum. After further washings, sections were mounted with antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).
Immunohistochemical stainings for EBV latent (EBNA2) and lytic (BZLF1) proteins and double immunofluorescence stainings for LMP1 and the B-cell marker CD79a and for BZLF1 and the plasma cell marker IgA, IgG, and IgM were performed in sections from paraformaldehyde (PFA)-fixed frozen brain tissue blocks of three MS donors analyzed in this study (MS79, MS92, and MS121), as previously described (19 (link), 35 (link)– (link)38 (link)). For double immunofluorescence staining for LMP1 and LMP2A, brain sections were incubated with anti-LMP2A rat MAb (1:50; clone TP4E11; Ascenion, Munich, Germany) and anti-LMP1 mouse MAb (1:100; clone CS.1-4; DakoCytomation) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin overnight at 4°C, and, after washing with a mixture of Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen, Eugene, OR) and tetramethylrhodamine (TRITC)-conjugated donkey anti-rat Ig (Jackson ImmunoResearch Laboratories, Cambridgeshire, UK), both diluted 1:300 in PBS containing 3% normal donkey serum. After further washings, sections were mounted with antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).
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