Six micrometers cryosections were obtained from skin biopsies and prepared on poly-L-lysine coated glass slides (Cytospin, Thermo Scientific). Staining for telomere-associated ɣH2AX foci (TAF) was then performed. Sections were stained for ɣH2AX (Ser139, Cell Signaling #9718, 1:250), p16INK4a (Abcam, ab108349, 1:100 or Sigma SAB5300499, 1:100) and HLA-E (clone 3D12, eBioscience, 1:100), followed by incubation with secondary antibody conjugated to various fluorochromes and washed in formamide/SSC prior to mounting with Vectorshield/DAPI (Vector Laboratories). Slides were air dried prior to hybridisation for 2 h with 40 pM PNA probe targeting the TelC telomeric repeat (Panagene, TelC Cy3, #14 1224PL-01). Imaging of TAF foci was then performed using a Leica SPE2 confocal microscope (Leica Microsystem). Imaging consisted of obtaining Z-stacks with a step-size of 0.5 µm. Analysis was performed using Fiji image analysis software (Fiji.sc).
Vectorshield dapi
Manufactured by Vector Laboratories
Sourced in United States
VectaShield/DAPI is a mounting medium and nuclear counterstain product offered by Vector Laboratories. It is designed to preserve fluorescence signals and provide nuclear staining in fluorescence microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Spe 2 confocal microscope, by Leica (3 mentions)
Cytospin, by Thermo Fisher Scientific (3 mentions)
Ab108349, by Merck Group (1 mentions)
Clone 3d12, by Thermo Fisher Scientific (1 mentions)
Telc cy3, by Panagene (1 mentions)
Fluorescent microscope, by Zeiss (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Cy3 anti mouse secondary antibody, by Jackson ImmunoResearch (1 mentions)
Streptavidin fitc, by Vector Laboratories (1 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
4 protocols using vectorshield dapi
1
Quantifying Telomere-Associated DNA Damage
2
Telomere Analysis of T Cell Subsets
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CD4+ and CD8+ T cells were isolated (as above) from HC and CL samples and prepared on poly-L-lysine coated glass slides by cytocentrifugation (Cytospin, Thermo Scientific, USA). Cytological specimens were then dried and fixed in ethanol/acetone prior to freezing. Specimens were thawed prior to staining. Specimens were PFA fixed, dehydrated in cold ethanol prior to permeabilization and blocking with BSA. Slides were washed prior to further dehydration across an ethanol gradient and air dried prior to hybridization with the PNA probe (Panagene, TelC Cy3, #14 1224PL-01) for 2 h in the dark as described in (16 (link)). Slides were subsequently washed in formamide/SSC prior to mounting with Vectorshield/DAPI (Vector Laboratories, USA). Imaging was performed using a Leica SPE2 confocal microscope using LAS X version 3.3.0 software (Leica Microsystem, Wetzlar, Germany). The images corresponded to a full z stack of CD4 or CD8 T cells were analyzed using ImageJ software.
3
Vinculin Immunofluorescence Staining
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Cells were then washed with warm 1×
phosphate-buffered saline (PBS) twice before being fixed with 4% formaldehyde
at room temperature for 15 min followed by permeabilization with a
solution of 0.1% Triton X-100 in PBS at room temperature for 5 min.
To block nonspecific binding, the samples were incubated in 1% bovine
serum albumin (BSA) in PBS for 1 h at room temperature. After blocking,
an anti-vinculin mouse primary antibody at a dilution of 1:500 in
1% BSA in PBS was added to the samples and incubated for 1 h at room
temperature. The samples were consequently washed with 0.5% Tween
20 in PBS three times for 5 min. A Cy3 anti-mouse secondary antibody
(1:200) (Jackson ImmunoResearch Laboratories Inc.), Phalloidin Alexa
Fluor 488 (1:100) (Thermofisher Scientific, U.K.) in 1% BSA in PBS,
was consequently added to the samples and incubated for 1 h at room
temperature followed by another three 5 min washes with 0.5% Tween
20 in PBS. The nuclei of the cells were stained using Vectorshield-DAPI
(Vector Laboratories) before being imaged with a fluorescent microscope
(Zeiss, GmbH, Germany). All reagents unless otherwise stated were
sourced from Sigma-Aldrich, U.K.
phosphate-buffered saline (PBS) twice before being fixed with 4% formaldehyde
at room temperature for 15 min followed by permeabilization with a
solution of 0.1% Triton X-100 in PBS at room temperature for 5 min.
To block nonspecific binding, the samples were incubated in 1% bovine
serum albumin (BSA) in PBS for 1 h at room temperature. After blocking,
an anti-vinculin mouse primary antibody at a dilution of 1:500 in
1% BSA in PBS was added to the samples and incubated for 1 h at room
temperature. The samples were consequently washed with 0.5% Tween
20 in PBS three times for 5 min. A Cy3 anti-mouse secondary antibody
(1:200) (Jackson ImmunoResearch Laboratories Inc.), Phalloidin Alexa
Fluor 488 (1:100) (Thermofisher Scientific, U.K.) in 1% BSA in PBS,
was consequently added to the samples and incubated for 1 h at room
temperature followed by another three 5 min washes with 0.5% Tween
20 in PBS. The nuclei of the cells were stained using Vectorshield-DAPI
(Vector Laboratories) before being imaged with a fluorescent microscope
(Zeiss, GmbH, Germany). All reagents unless otherwise stated were
sourced from Sigma-Aldrich, U.K.
4
Quantifying Telomeric DNA Damage
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Flow-sorted CD56dim and CD56neg NK cells were cytocentrifuged onto poly-L-lysine coated glass slides (Cytospin, Thermo Scientific). Staining for telomere-associated γH2A.X foci (TAF) was then performed as previously described [42 (link)]. Slides were air dried prior to hybridisation with 40 pM PNA probe targeting the TelC telomeric repeat (Panagene, TelC Cy3, #14 1224PL-01) for 2 hours. Sections were then counter stained for γH2A.X (Ser139, Cell Signaling #9718), followed by incubation with biotinylated secondary antibody (BA-1000, Vector) and FITC-streptavidin (A-2011). Slides were subsequently washed in formamide/SSC buffer prior to mounting with Vectorshield/DAPI (Vector Laboratories). Imaging was performed using a Leica SPE2 confocal microscope (Leica Microsystem). Analysis was performed using Fiji image analysis software (Fiji.sc).
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