Paraffin-embedded dorsal skin sections were deparaffinized, rehydrated, and antigen was retrieved by incubating sections in boiling in sodium citrate buffer (10 mM, pH 6) for 15 min. Slides were then incubated with primary antibodies: rabbit anti-α-SMA (Abcam, Cat. ab5694, dilution 1:600) or rabbit anti-COL1A1 (Abcam, Cat. ab7778, dilution 1:600) at 4°C for 10 h. After incubation, sections were washed with 0.1 M PBS and incubated with goat anti-rabbit IgG-Alexa Fluor 488 (Abcam, Cat. ab150077, dilution 1:1000) or goat anti-rabbit IgG-Alexa Fluor 555 (Abcam, Cat. ab150078, dilution 1:1000) at room temperature for 1 h. After an additional rinse, sections were mounted by VECTASHIELD mounting medium with DAPI (Vector Lab, Shanghai, China) and observed under confocal microscopy (Olympus, Japan) (Li et al., 2021a (link)). The fluorescence intensity was measured on fields (460 μm × 460 μm) around the wound area using the ImageJ software (Li et al., 2021a (link)). At least six replicates were analyzed for each treatment.
Alexa fluor 555 goat anti rabbit igg
Manufactured by Abcam
Sourced in United States
Alexa Fluor 555 goat anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 555 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti rabbit igg, by Abcam (3 mentions)
Alexa fluor 488 goat anti mouse igg, by Abcam (3 mentions)
Ab150077, by Abcam (2 mentions)
Rabbit anti col1a1, by Abcam (1 mentions)
Rabbit anti α sma, by Abcam (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Confocal microscopy, by Olympus (1 mentions)
Anti myosin ld, by Santa Cruz Biotechnology (1 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica camera (1 mentions)
Anti claudin 5, by Abcam (1 mentions)
Anti occludin, by Abcam (1 mentions)
Anti gfap, by Novus Biologicals (1 mentions)
Phalloidin ifluor 488 reagent, by Abcam (1 mentions)
Anti cd31, by Abcam (1 mentions)
Goat serum, by Merck Group (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Mouse anti map2ab, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Prolong diamond mounting medium, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
9 protocols using alexa fluor 555 goat anti rabbit igg
1
Immunofluorescence Analysis of Skin Wound Healing
2
Immunofluorescence Staining of Cellular Markers
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Cells were washed with PBS three times, fixed for 15 min in 4% PFA, permeabilized with 0.5% Triton X-100 for 15 min, blocked in 10% goat serum for 1 h at RT, and incubated with primary antibodies overnight at 4°C. The primary antibodies used were as follows: anti-Myosin ld (1:100, Santa Cruz Biotechnology, sc-515292), anti-CD31 (1:100, abcam, ab222783), anti-Occludin (1:100, abcam, ab216327), anti-Claudin-5 (1:100, abcam, ab131259), anti-ZO-1 (1:100, abcam, ab276131), anti-PDGFR-β (1:200, Invitrogen, 14-1402-82), and anti-GFAP (1:100, Novus, NBP1-05197). The following day, the cells were incubated with secondary antibodies for 1 h at RT. The secondary antibodies used were as follows: Goat Anti-Rabbit IgG Alexa Fluor® 555 (1:1000, abcam, ab150078) and Goat Anti-mouse IgG Alexa Fluor®488 (1:1000, abcam, ab150113). After washing, the cells were incubated with DAPI or Phalloidin-iFluor 488 Reagent (1:1000, abcam, ab176753) for 15 min at RT. Images were captured using a LEICA TCS SP8 laser scanning confocal microscope. Colocalization analysis of Myo1d and F-actin was performed using Fiji, and Pearson’s R-value was calculated to quantify pixel intensity correlations.
3
Immunofluorescence Staining of Cultured Cells
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Cells cultured on coverslips in 24-well plates (Costar) were fixed in 4% paraformaldehyde (PFA, Sigma) for 20 min at RT, washed twice in 0.15 M Sørensen’s buffer for 15 min and then permeabilized by washing three times in 0.1% Triton X-100 (Sigma) in 0.05 M TBS for 15 min at RT, followed by blocking in 5% goat serum (Millipore) in 0.05 M TBS for 30 min at RT. Afterwards, primary antibodies diluted in 5% goat serum were added to the cells in the following concentrations: rabbit anti-TH (Millipore #AB152) 1:600, mouse anti-MAP2a + b (Sigma #M1406) 1:2000, rabbit anti-LAMP1 (Abcam #108597) 1:1000, rabbit anti-TOM20 (Santa-Cruz #SC-11415) 1:1000. The cells were washed three times in 0.1% Triton X-100 in 0.05 M TBS for 15 min at RT and incubated with fluorophore-conjugated secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen #A11008), Alexa Fluor 488 goat anti-mouse IgG (Invitrogen #A11001), Alexa Fluor 555 goat anti-rabbit IgG (Abcam #150078) or Alexa Fluor 555 goat anti-mouse IgG (Molecular probes #21422) 1:500 diluted in 5% goat serum in 0.05 M TBS for 2 h at RT. The cells were washed twice in 0.05 M TBS for 15 min at RT and then counterstained with 10 µM DAPI (Sigma) for 15 min at RT to stain all nuclei. Finally, cells were mounted onto glass slides using ProLong® Diamond mounting medium (Molecular Probes).
4
Immunofluorescence Staining of Cell Markers
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Immunofluorescence staining was performed according to our previous protocol24 (link). Briefly, the cells were incubated with primary antibodies: monoclonal mouse antibody to PHGDH (1:50, Santa Cruz Biotechnology) and SHMT2 (1:80, Abcam), and monoclonal rabbit antibodies to PSPH (1:50, Santa Cruz Biotechnology), PSAT (1:100, Novus), SHMT1 (1:80, Novus), TOM20 (1:100, Abcam), and Ki-67 (1:400, Abcam) in a humidified atmosphere for 1 hour at 37 °C. As secondary antibodies, Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 555 goat anti-rabbit IgG were used (both; 1:200, Abcam). Nuclei were stained with Hoechst (50 μg/ml). Images were captured with the 40X oil objective lens on the Olympus Provis fluorescence microscope (Olympus Optical, Tokyo, Japan).
5
Lung Tissue Immunofluorescence Staining Protocol
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After drying overnight at 37°C, the 5 μm lung tissue sections were deparaffinized using a gradient series of xylene and ethanol. Antigen retrieval was performed by microwave heating the sections for 20 min in 10 mM citric acid buffer (pH 6.0). After three washes with 1x PBS, the sections were blocked using 10% goat serum albumin for 1 h. KLF2 (1 : 100, A16480, ABclonal, Wuhan, China) and Ki67 (1 : 100, A2094, ABclonal, Wuhan, China) were diluted in 10% goat serum albumin, and 30 μL was added to the sections (overnight at 4°C). The following day, the sections were incubated at room temperature for 2 h with Alexa Fluor-488 goat anti-rabbit IgG (1 : 200; AB150077; Abcam) and Alexa Fluor-555 goat anti-rabbit IgG (1 : 200; AB150078; Abcam). Finally, the sections were treated with a mounting medium containing 4′,6-diamidino-2-phenylindole (Solarbio, Beijing, China) and the images were obtained using a scanning microscope (C1; Nikon, Tokyo, Japan).
6
Immunofluorescence Labeling of Epithelial-Mesenchymal Markers
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Cultured cells grown on coverslips in a 24-well plate were fixed with 2% paraformaldehyde (Sigma-Aldrich) for 30 min, washed in PBS, and then permeabilized with 0.1% Triton-X-100 (Fisher Scientific, Pittsburgh, PA) made in PBS for 15 min. Following a PBS wash, non-specific proteins were blocked in 2% BSA for 15 min at RT. The cells were incubated with a mixture of two primary antibodies: monoclonal rabbit antibody to vimentin (1:100, ab92547, Abcam) and monoclonal mouse antibody to E-cadherin (1:50, ab1416, Abcam) in a humidified atmosphere for 1 hour at 37°C. Coverslips were then probed with Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 555 goat anti-rabbit IgG (both; 1:200, Abcam) in the dark for 15 min at RT. Following a PBS wash, nuclei were stained with Hoechst 33342 (50 μg/ml) for 5 min at RT, washed and mounted in an aqueous-based mounting medium Clearmount™ (Invitrogen). Images were captured with the 40X oil objective lens on the Olympus Provis fluorescence microscope (Olympus Optical).
7
Multiparameter Immunophenotyping of Neutrophils
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Anti-mouse Ly6G (IA8; 127608; BioLegend, CA, USA), anti-mouse CD11b (M1/70; 101212; BioLegend), anti-mouse CD63 (NVG-2; 143920; BioLegend), anti-mouse CXCR2 (SA044G4; 149315; BioLegend), anti-mouse CXCR4 (L276F12; 146511; BioLegend), and anti-mouse CD4 (GK1.5; 100406; BioLegend) antibodies were used to stain the surface molecules of samples. For intracellular staining, anti-mouse IFN-γ (XMG1.2; 505809; BioLegend) and anti-mouse IL-17A (TC11-18H10.1; 506903; BioLegend) antibodies were used. For NET components staining, anti-mouse MPO (ab90812; Abcam, MA, USA), anti-mouse CitH3 (ab5103; Abcam), anti-mouse PAD4 (ab96758; Abcam), anti-mouse NE (ab38672; Abcam), anti-mouse β-actin (8457S; Cell Signaling Technology, MA, USA), Alexa Fluor 488 goat anti-rabbit IgG (ab150077; Abcam), Alexa Fluor 555 goat anti-rabbit IgG (ab150078; Abcam), and Alexa Fluor 647 donkey anti-rabbit IgG (101,231; BioLegend) antibodies were used.
8
Immunofluorescence Analysis of ROCK1
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5 × 104 cells were seeded into slides (Millipore, MA, USA) and fixed with 4% paraformaldehyde (PFA) for 30 min. PBS was used to rinse the slides for 3 times, and 5% BSA was applied to block the slides for 1 h at 37℃ and primary antibodies were used to incubate the slides at 4 °C overnight. Next day, slides were rinsed with PBS for 3 times and incubated with secondary antibodies in the dark at 37℃ for 1 h. Primary antibody included Anti-ROCK1 (Cell Signaling Technology) and Alexa Fluor® 488 goat (Abcam, Cambridge, MA, USA) and secondary antibody included Alexa Fluor® 555 goat anti-rabbit IgG. DAPI was used to visualize the nuclei of cells in the dark for 5 min. Analyzing slides by fluorescent microscopy (10x).
9
Immunofluorescence Analysis of COX-2 and iNOS
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HTR-8/SVneo cells (50 × 10 3 ) were grown to confluence on glass coverslips and incubated with LPA 10 μM for 6 h. Cells were fixed (10 min, room temperature, 3.7% w/v paraformaldehyde) and permeabilized with 0.1% TRITON X-100 in PBS for 5 min. Non-specific binding sites were blocked (60 min, 40 mg/ mL BSA in PBS). Slices were incubated overnight at 4°C with anti-COX-2 (1:100, mouse polyclonal antibody, catalog # 160126, Cayman Chemical) or anti-iNOS (1:100, polyclonal antibody, catalog # 160826, Cayman Chemical). Afterwards, cells were incubated for 60 min at room temperature with Alexa-Fluor 555 goat anti-rabbit IgG (1:500, Abcam, Tecnolab, Buenos Aires, Argentina). Immunoreactive specificity was assessed by omitting the first antibody. Finally, cells were mounted on glass-slices using DABCO mounting medium (Sigma, Buenos Aires, Argentina) and observed by fluorescence microscopy at 100x (Nikon Eclipse 200).
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