Cell seeded on glass coverslips were fixed with 2% (v/v) PFA in PBS at room temperature for 15 min and then washed three times in PBS and once in PBS with 50 mM glycine. Dilutions of primary and secondary antibodies were prepared in buffer A: PBS with 0.2% (w/v) Bovine Serum Albumin (BSA) and 0.1% (w/v) saponin. The coverslips were incubated with the primary antibodies for 1 h at room temperature after being washed once in buffer A. Coverslips were washed three times in buffer A, followed by incubation with secondary antibodies and phalloidin for 45 min if necessary. The final washing steps were performed three times in buffer A, once in PBS, and once in water. The coverslips were mounted on glass slides using ProLong™ Gold Antifade Mount with DAPI (Thermo Fisher Scientific).
Prolong gold antifade mount with dapi
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Prolong Gold Antifade Mount with DAPI is a mounting medium designed to preserve fluorescence signals in microscopy samples. It contains an antifade agent to protect against photobleaching and DAPI, a fluorescent dye that binds to DNA, enabling visualization of nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Axiovision 4, by Zeiss (4 mentions)
Axiovert 200 fluorescence microscope, by Zeiss (3 mentions)
Peptidylprolyl isomerase b ppib, by Advanced Cell Diagnostics (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Axio observer 3, by Zeiss (2 mentions)
Anti alkaline phosphatase alp antibody, by R&D Systems (2 mentions)
Leukocyte tartrate resistant acid phosphatase staining kit, by Merck Group (2 mentions)
Avidin biotin complex, by Vector Laboratories (2 mentions)
Alexa fluor 488 568 conjugated secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Deltavisioncore system, by Olympus (1 mentions)
Applied precision s softworx software, by GE Healthcare (1 mentions)
α gfp, by Abcam (1 mentions)
Anti krt10, by Abcam (1 mentions)
Anti krt14, by Thermo Fisher Scientific (1 mentions)
Anti krt5, by Abcam (1 mentions)
Anti cd200, by Abcam (1 mentions)
Anti ki67, by Abcam (1 mentions)
Fluoview fv3000 confocal microscope, by Olympus (1 mentions)
26 protocols using prolong gold antifade mount with dapi
1
Immunofluorescence Staining on Coverslips
2
Immunofluorescence Staining on Cell Monolayers
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Cell monolayers seeded on glass coverslips were fixed with 4% (v/v) paraformaldehyde in PBS at room temperature for 15 min, then washed three times in PBS and once in PBS containing 50 mM glycine. Primary and secondary antibody dilutions were prepared in the buffer A: PBS containing 0.2% (w/v) BSA and 0.1% (w/v) saponin. The coverslips were washed once in buffer A and then incubated for 1 h at room temperature (RT) with the primary antibodies. Following one wash step in buffer A, the coverslips were incubated for 30 min at RT with the secondary antibodies. For phalloidin staining, the coverslips were washed in buffer A and incubated overnight in the same buffer with phalloidin at 4 °C. The final wash step was done once in buffer A, once in PBS and once in water. The coverslips were mounted onto glass slides using ProLong™ Gold Antifade Mount with DAPI (ThermoFischer Scientific). Images were acquired on an Applied Precision DeltavisionCORE system (unless stated otherwise), mounted on an Olympus inverted microscope, equipped with a CoolSnap HQ2 camera (Photometrics), using the 40×1.3 NA UPLFLN or the 60×1.42 NA PLAPON-PH oil immersion objectives. Images were deconvolved with Applied Precision’s softWorx software (GE Healthcare).
3
RNAscope Analysis of Intestinal Cell Markers
4
Immunohistochemical Analysis of Tissue Sections
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Tissue was fixed with 4% paraformaldehyde then frozen in Optimal Cutting Temperature Compound (OCT) and sectioned at 20 µM. Sections were blocked with IHC/ICC Blocking Buffer (Invitrogen) with 0.4% Triton X-100 (Sigma), incubated with primary and secondary (1:5000) antibodies and finally mounted in Prolong Gold Antifade Mount with DAPI (ThermoFisher). Antibodies and dilutions Anti-Ki67, 1:100 (Abcam ab15580); Anti-KRT14 1:200 (Thermo Fisher MA1-06323); Anti-CD200, 1:50 (ls-b11638); AQ3 1:200 (Abcam ab125219); Anti-KRT10 1:200 (Abcam ab9025); Anti-KRT40, 1:100 (Abcam ab16113); Anti-KRT5, 1:100 (Abcam ab64081). Immunostained samples were visualized by confocal microscopy (Olympus Fluoview FV3000 Confocal Microscope). Line profile intensity was measured using ImageJ (NIH).
5
Immunofluorescence Staining of Cells
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Cells were grown on 18 mm coverslips (VWR, 48380-046) within 12-well plates and allowed to adhere for 48 hr. Coverslips were washed twice with PBS and then fixed with 4% paraformaldehyde (Thermo Scientific, 28908, lot RK2302081) for 15 min at room temperature; cells were then washed trice with PBS containing 10mM of glycine and either permeabilized with 0.1% Triton-X 100 (Calbiochem, 9410, lot UB16DZEMS) in PBS for 5 min, or left intact. Cells were subsequently washed three times with PBS and blocked in 3% milk for 1 hour at room temperature. Cells were incubated with primary antibodies (1:100 in blocking buffer) overnight at 4°C and washed twice prior to incubation with secondary antibodies (1:200 in blocking buffer) for 1 hour at room temperature. Cells were then washed twice and mounted with ProLong Gold Antifade Mount with DAPI (Thermo Scientific, P36931, lot 1836709). Images were taken on a Leica SP5 confocal microscope with a 63× glycerol immersion lens.
6
Podocyte Actin Cytoskeleton Visualization
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Podocytes were seeded onto coverslips in a 12-well plate and following differentiation were treated with cytokines as described above for 1 h and 24 h. Additionally, podocytes were treated with 10% sera from patients with active LN by British Isles Lupus Assessment Group (renal BILAG A/B), inactive LN (renal BILAG D/E) or age- and sex-matched healthy controls for 1 h. These cells were then fixed in 4% formalin in PBS, permeabilized using PBS with 0.1% Triton X-100, and the actin cytoskeleton was stained with Alexa Fluor 488 phalloidin (Thermo Fisher Scientific). The coverslips were mounted using ProLong gold antifade mount with DAPI (Thermo Fisher Scientific). Images were taken on a Leica TCS SPE confocal microscope, and five random images per coverslip were collected in which a minimum of three nuclei were in each field of view. ImageJ software was used to analyze the area of phalloidin stain per field of view. Briefly, a threshold was set on the 488-nm channel, and the software determined the number of pixels above this threshold and the area.
7
RNA-FISH for LGR5 Expression Across Species
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RNA in situ hybridization (RNA-FISH) was performed using RNAscope (Advanced Cell Diagnostics) according to the manufacturer's instructions. Briefly, paraffin embedded skin tissue was sectioned at 7um. Slides were deparaffinized with xylene, then heat treated followed by protease digestion. The tissue was hybridized with a 10 ZZ probe targeting either the 560–1589 region of Homo sapiens LGR5 mRNA, the 466–1464 region of Sus scrofa LGR5 mRNA, or the 494–1423 region of Macacca mulatta LGR5 mRNA. As controls, a positive control probe was used against porcine (1–642 region) or human (139–989 region) cyclophilin B, Rhesus peptidyprolyl isomerase B (119–916 region), or negative control probes targeting the bacterial gene dapB were used, followed by chromogenic development. Slides were washed, then mounted with Prolong Gold Antifade Mount with DAPI (ThermoFisher) and imaged by confocal microscopy.
8
Visualizing Neutrophil Granular Proteins
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To detect neutrophil granular proteins, human or mouse neutrophils (250,000 cells in 0.5 ml) were left to adhere onto 13 mm coverslips for 1 h and subsequently treated with P. aeruginosa strains for 2 h at 37°C at multiplicity of infection (MOI) 10. Samples were fixed with 4% paraformaldehyde, washed, and blocked using 5% donkey serum (Sigma Aldrich). BPI was detected using mouse anti-BPI antibody (1:200, Santa Cruz Biotechnology, Inc.) for human neutrophils or rabbit anti-mouse BPI antibody (1:200, ABclonal), followed by donkey anti-mouse Alexa Fluor 488 secondary antibody or donkey anti-rabbit Alexa Fluor 647 (1:500, Jackson ImmunoResearch). Neutrophil elastase (NE) was detected using rabbit anti-NE IgG (Abcam), followed by donkey anti-rabbit Alexa Fluor 647 secondary antibody (1:500, Jackson ImmunoResearch). Myeloperoxidase (MPO) was detected using rabbit anti-MPO IgG (Cell Signaling), followed by donkey anti-rabbit Alexa Fluor 647 secondary antibody (1:500, Jackson ImmunoResearch). Samples were mounted using ProLong Gold Antifade Mount with DAPI (ThermoFisher Scientific) and visualized with the laser point scanning confocal microscope (LSM 510 META, Zeiss), 63X.
9
Immunolabeling of Mouse IgA Antibodies
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Four-micrometer-thick paraffin sections were deparaffinized, rehydrated, and permeabilized with Triton X-100 0.01% in PBS for 1 h at room temperature, blocked with 1% BSA in PBS for 1 h, then stained with 2.5 μg/mL anti-mouse IgA primary antibody (556969; BD Pharmingen, NJ, USA) in blocking buffer (1% BSA in PBS) for 18 h at 4 °C. Sections were subsequently incubated with 2 μg/mL Alexa Fluor 594 goat anti-rat IgG (H + L) secondary antibody (A-11007, Thermo Fisher Scientific, MA, USA) for 2 h at 4 °C. After washing, specimens were mounted with ProLong™ Gold Antifade Mount with DAPI (P36935, Thermo Fisher Scientific, MA, USA) and stored at 4 °C. Image acquisition was performed with a Zeiss LSM780 confocal microscope (Zeiss, Jena, Germany) equipped with a 40×/1.3 NA EC Plan-Neofluar oil objective. High-resolution images of the whole sample were taken through a tile scan.
10
Multispecies mRNA Detection by RNAscope Histology
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Fluorescent in situ hybridization to detect mRNA targets (RNAscope, Advanced Cell Diagnostics) was performed in accordance with the manufacturer's instructions. Briefly, formalin‐fixed paraffin embedded intestinal tissue from human, mouse, or pig was sectioned at 7 μm. Slides were deparaffinized with xylene, then heat‐induced epitope retrieval treated followed by protease digestion. The tissue was hybridized with the species‐specific probes targeting LGR5, HOPX, SOX9, LYZ, and OLFM4 mRNA or a positive control probe peptidylprolyl isomerase B (PPIB) (Advanced Cell Diagnostics), followed by chromogenic or Tyramide Signal Amplification technology development per user manual. Porcine tissue slides were then incubated in primary antibody α‐GFP (abcam catalogue # ab183734), diluted (1:50) followed by secondary antibody (Alexa Fluor 488). Slides were washed, then mounted with Prolong Gold Antifade Mount with DAPI (Thermo Fisher) and imaged by confocal microscopy.
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