Immunohistochemical Staining performed after being dewaxed of mice tissue sections. Sections permeabilized with 0.1% Triton X-100, and blocked with 10% goat serum at room temperature for 1 h. Samples were incubated with anti-rabbit SOCS3 antibody (for;1:500, ab16030, Abcam, USA), anti-rabbit MMP9 (1:500, ab76003, Abcam, Cambridge, MA, USA), anti-rabbit-AQP9 antibody (1:600, ab84828, Abcam, Cambridge, MA, USA), and anti-rabbit-MPO antibody (1:600, EPR20257, Abcam, Cambridge, MA, USA) at 4°C overnight. On the 2nd day, slides were incubated with goat anti-rabbit/rabbit FITC secondary antibody for 1h at room temperature. The nuclei were dyed by 4′,6-diamidino-2-phenylindole (DAPI) for 5min (4′,6-diamidino-2-phenylindole, Vector, ZsBio, Beijing, China). Images were obtained using Zeiss fluorescence microscope (Germany) and Nikon microscope (Nikon, Tokyo, Japan). At least five fields for each coverslip were captured. The number of cells, antigen distribution, and cellular fluorescence intensity that represented antigenicity were measured using the captured images. The image analysis was performed with ImageJ (NIH, Bethesda, MD, USA).
4 6 diamidino 2 phenylindole (dapi)
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom, Canada, Germany, Japan, China, Spain, France, Colombia
DAPI is a fluorescent dye used for staining and visualizing DNA in biological samples. It binds to the minor groove of DNA, emitting blue fluorescence when excited by ultraviolet or violet light. DAPI is commonly used in fluorescence microscopy and flow cytometry applications.
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Lab products found in correlation
Vectashield mounting medium, by Vector Laboratories (162 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (123 mentions)
Lsm 700, by Zeiss (84 mentions)
Triton x 100, by Merck Group (70 mentions)
Vectashield, by Vector Laboratories (57 mentions)
Bovine serum albumin (bsa), by Merck Group (57 mentions)
Lsm 710 confocal microscope, by Zeiss (52 mentions)
Paraformaldehyde (pfa), by Merck Group (51 mentions)
Lsm 700 confocal microscope, by Zeiss (40 mentions)
Mounting medium, by Vector Laboratories (38 mentions)
Lsm 510, by Zeiss (37 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (36 mentions)
In situ cell death detection kit, by Roche (32 mentions)
Lsm 710, by Zeiss (32 mentions)
Fluorescence microscope, by Olympus (31 mentions)
Lsm 780 confocal microscope, by Zeiss (29 mentions)
Lsm 780, by Zeiss (27 mentions)
Lsm 880 confocal microscope, by Zeiss (23 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (21 mentions)
Vectashield mounting media, by Vector Laboratories (20 mentions)
2 775 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Immunohistochemistry Analysis of Mice Tissues
2
Immunohistochemical Analysis of Tissue Markers
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We used a Tris‐EDTA buffer (pH 9.0) antigen retrieval method (for 10 minutes at 110°C) for all immunohistochemical staining. The primary antibodies used were as follows: anti‐p53 mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐active caspase‐3 rabbit polyclonal antibody (Cell Signaling Technology, Danvers, MA, USA), anti‐podoplanin hamster monoclonal antibody (AngioBio, Del Mar, CA, USA) and anti‐CD31 rabbit polyclonal antibody (Lab Vision, Fremont, CA, USA). We incubated the slides with corresponding biotinylated secondary antibodies using an established labeled streptavidin‐biotin method (Dako, Glostrup, Denmark), with exposure to diaminobenzidine and hematoxylin counterstain for visualizing the immunocomplexes formed. In addition, anti‐phospho‐Akt‐Ser473/Thr308 rabbit polyclonal antibody (Cell Signaling Technology) and anti‐rabbit Alexa‐594 antibody (Thermo Fisher Scientific, Waltham, MA, USA) were used for immunofluorescence staining. Nuclear staining was conducted with DAPI (Vector Labs, Burlingame, CA, USA); phospho‐Akt‐positive areas and viable regions with DAPI‐positive nuclei in the same field were digitally captured at 40× magnification. These digitized images were measured using the ImageJ program (NIHR public domain) and the results are expressed as a percentage of phospho‐Akt‐positive areas to nuclear‐positive areas.
3
Quantifying Keratinocyte Proliferation and Differentiation
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Formalin-fixed cryosections (10 µM-thickness) were incubated with primary antibodies against Ki-67 (Abcam; 1:100), Keratin 16 (Abcam; 1:250), Keratin 17 (Abcam; 1:100) and CD49f (BD Pharmingen; 1:100) overnight followed by application of the corresponding Alexa-546 or Alexa-555-labeled antibodies (Invitrogen, UK) for 45 min at 37 °C. Cell nuclei were counterstained with DAPI (Vector Labs, UK). Image analysis was performed using a fluorescent microscope in combination with DS-C1 digital camera and ACT-2U image analysis software (Nikon).
For BrdU analysis, the keratinocytes were seeded on collagen-coated sterile glass coverslips in a 6-well cell culture dish and transfected with miR-200c inhibitor or negative control RNA. 48 h after treatment, cells were treated with 10 µM BrdU (Sigma; 2 hours; 37 °C). Next, the cells were fixed with 4% paraformaldehyde (30 min, RT) followed by denaturation in 2 M HCl (30 min, 37 °C) and neutralisation in 0.1 M sodium borate. The cells were stained with FITC-conjugated Anti-BrdU (BD Biosciences; 30 min). Cell nuclei were counterstained with DAPI (Vector Labs, UK). Fluorescent microscopy images from 10 randomly selected fields per coverslip were taken, and the numbers of DAPI+ nuclei and FITC+ nuclei were counted using ImageJ software (NIH, Bethesda, MD USA). Statistical analysis was performed using Wilcoxon rank sum test.
For BrdU analysis, the keratinocytes were seeded on collagen-coated sterile glass coverslips in a 6-well cell culture dish and transfected with miR-200c inhibitor or negative control RNA. 48 h after treatment, cells were treated with 10 µM BrdU (Sigma; 2 hours; 37 °C). Next, the cells were fixed with 4% paraformaldehyde (30 min, RT) followed by denaturation in 2 M HCl (30 min, 37 °C) and neutralisation in 0.1 M sodium borate. The cells were stained with FITC-conjugated Anti-BrdU (BD Biosciences; 30 min). Cell nuclei were counterstained with DAPI (Vector Labs, UK). Fluorescent microscopy images from 10 randomly selected fields per coverslip were taken, and the numbers of DAPI+ nuclei and FITC+ nuclei were counted using ImageJ software (NIH, Bethesda, MD USA). Statistical analysis was performed using Wilcoxon rank sum test.
4
Immunofluorescence Assay for GDF15 and Actin
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The immunofluorescence assay was performed according to a previously reported protocol (27). Briefly, cells were seeded in a chamber slide (Lab-Tek II; Thermo Fisher Scientific) and incubated, fixed with 10% formalin, permeabilized with 0.5% Triton X-100, and then blocked in blocking buffer (4% BSA in PBS). Cells were stained with anti-GDF15 antibody, washed with PBS, and treated with a secondary antibody. Finally, nuclei were counterstained with DAPI (Vector Laboratories, Burlingame, CA, USA), and coverslips were mounted on the slides. To analyze the actin cytoskeleton, cells were stained with rhodamine-conjugated phalloidin (Sigma) and washed three times with PBS. The nuclei were stained with DAPI (Vector Laboratories).
5
Apoptosis Detection in Lung Cancer Cells
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TUNEL assay was performed by using the DeadEndTM Fluorometric TUNEL System (Promega, Madison, Wisconsin, USA) for in situ detection of apoptotic cells, according to the manufacturer's instructions. Lung cancer cells (2 × 104 cells/well) were cultured on 8-chamber slides, after cells were treated with SVT. The cells were washed with PBS and fixed by incubation in 4% paraformaldehyde in PBS for 20 min at room temperature. Membrane was permeabilized by exposure to 0.1% Triton X-100 in PBS for 5 min at room temperature. For DAPI staining, slides were incubated for 15 min at room temperature in the dark with a mounting medium for fluorescence containing DAPI (Vector Laboratories, Inc., Burlingame, CA). The cells were then observed through a fluorescence microscope (Leica Microsystems AG, Wetzlar, Germany). The total number of cells in a given area was determined by using DAPI and TUNEL staining. The apoptotic index was determined as the number of DAPI-stained TUNEL-positive cells divided by the total number of cells counted × 100.
6
Localization of Transfected Cells
7
Localization of Transfected Cells
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For localization studies, MPT cells were plated on glass coverslips and fed with
Opti-MEM medium (Invitrogen, Grand Island, NY, USA) supplemented with 5% FBS in
5% CO2 at 37°C. Cells were transfected with 0.8
μg of plasmid DNA with LIPOFECTAMINE 2000 for 4 h. Transfected
cells were stabilized for 48 h before fixation with 4% paraformaldehyde in PBS and
rinsed in PBS, prior to applying mounting solution with DAPI (Vector Laboratories,
Burlingame, CA, USA). For F-actin staining, cells were fixed with 4%
paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100, stained with
Rhodamine-phalloidin (Molecular Probes Eugene, OR, USA) and mounted with DAPI (Vector
Laboratories, Burlingame, CA, USA). Images were acquired using an inverted fluorescent
microscope with a 40x objective (Olympus) and OpenLab software (Improvision).
Opti-MEM medium (Invitrogen, Grand Island, NY, USA) supplemented with 5% FBS in
5% CO2 at 37°C. Cells were transfected with 0.8
μg of plasmid DNA with LIPOFECTAMINE 2000 for 4 h. Transfected
cells were stabilized for 48 h before fixation with 4% paraformaldehyde in PBS and
rinsed in PBS, prior to applying mounting solution with DAPI (Vector Laboratories,
Burlingame, CA, USA). For F-actin staining, cells were fixed with 4%
paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100, stained with
Rhodamine-phalloidin (Molecular Probes Eugene, OR, USA) and mounted with DAPI (Vector
Laboratories, Burlingame, CA, USA). Images were acquired using an inverted fluorescent
microscope with a 40x objective (Olympus) and OpenLab software (Improvision).
8
Apoptosis Assessment in IVF Blastocysts
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Blastocysts that developed after RR or histamine treatment during IVF were fixed in 4% paraformaldehyde in PBS for detection of apoptosis by terminal deoxynucleotidyl
transferase-mediated dUDP nick-end labeling (TUNEL) using an In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.
Blastocysts were permeabilized using 0.5% (v/v) Triton X-100 for 30 min at 4°C. The fixed blastocysts were incubated in TUNEL reaction medium for 1 h at 38.5°C, then washed three
times with 0.1% PVA-PBS and mounted on slides with mounting solution containing 1.5 μg/ml of 4’,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA).
DAPI-labeled or TUNEL-positive nuclei were examined using the forementioned iRiSTM digital cell imaging system following TUNEL assay and DAPI staining, and the number of
apoptotic nuclei and total number of nuclei were counted.
transferase-mediated dUDP nick-end labeling (TUNEL) using an In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.
Blastocysts were permeabilized using 0.5% (v/v) Triton X-100 for 30 min at 4°C. The fixed blastocysts were incubated in TUNEL reaction medium for 1 h at 38.5°C, then washed three
times with 0.1% PVA-PBS and mounted on slides with mounting solution containing 1.5 μg/ml of 4’,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA).
DAPI-labeled or TUNEL-positive nuclei were examined using the forementioned iRiSTM digital cell imaging system following TUNEL assay and DAPI staining, and the number of
apoptotic nuclei and total number of nuclei were counted.
9
Immunostaining Protocol for Brain and Cell Cultures
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For brain sections: sections from the entire brain were washed in PBS-0.2% Triton and blocked with goat serum (1:100 in PBS, Vector) for 60 min. Incubation with a primary antibody in PBS-0.2% Triton was performed overnight at 4°C. After several washes, the primary antibody against the second antigen was added in PBS-0.2% Triton, and the sections were again incubated at 4°C overnight. Incubation with the two Alexa Fluor secondary antibodies (1:1000 in PBS) was performed for 60 min at room temperature. The slides were mounted with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI) to label the nuclei (Vector). For cell cultures: sections were rinsed once in 50 mmol/L NH4Cl, and cells were permeabilised with Triton X-100 (0.1%, 10 min at room temperature). Subsequently, the slides were incubated with primary antibodies at 4°C overnight, and labelling was performed by a reaction with the appropriate Alexa Fluor secondary antibodies (1:400-Invitrogen) for 45 min at room temperature. The cells were mounted with Vectashield medium containing DAPI (Vector, Burlingame, USA).
10
Cellular Proliferation and Differentiation Assays
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Proliferation and differentiation were analysed as described [68 (link), 69 ]. Briefly, proliferation was measured using 5-bromo-2’-deoxy-uridine (BrdU) labelling (Roche, Basel, Switzerland). Cells were pulsed with 10 μM BrdU, fixed and incubated with anti-BrdU antibody (1:100) followed by anti-mouse Ig-fluorescein antibody (1:200) and mounted onto a glass slide using DAPI (Vectashield, Vector Laboratories, CA, USA). Images were captured using fluorescence microscope BX53 (Olympus Corporation, Shinjuku, Tokyo, Japan) at ×40 magnification.
For differentiation, cells were cultured in differentiation media consisting of either basal DMEM or RPMI 1640 with 2% Horse Serum (HyClone, Cytiva, U.S.) for 2–5 days. Cells were incubated with anti-MHC primary antibody (MHC; R&D Systems, Minneapolis, MN, USA) (1:400) followed by secondary goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (Thermo Fisher scientific). Coverslips were mounted with DAPI (Vectashield, Vector Laboratories, CA, USA) and imaged with BX53 microscope (Olympus Corporation) at ×40 magnification.
For differentiation, cells were cultured in differentiation media consisting of either basal DMEM or RPMI 1640 with 2% Horse Serum (HyClone, Cytiva, U.S.) for 2–5 days. Cells were incubated with anti-MHC primary antibody (MHC; R&D Systems, Minneapolis, MN, USA) (1:400) followed by secondary goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (Thermo Fisher scientific). Coverslips were mounted with DAPI (Vectashield, Vector Laboratories, CA, USA) and imaged with BX53 microscope (Olympus Corporation) at ×40 magnification.
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