To avoid skin contraction, complete wound specimens and the normal skin tissue within 2–3 mm around the wounds were collected on days 3 and 21 post-operation. The skin tissues were fixed overnight by 4% polyoxymethylene and then dehydrated by 20% sucrose. After embedding in optimal cutting temperature compound and freezing in liquid nitrogen, the specimens were cut into 5-μm sections for immunostaining. After blocking with 5% BSA, skin sections were incubated with a mouse anti-CD31 antibody (Abcam, Cambridge, MA, USA) or anti-CD68 antibody (Abcam), followed by a goat anti-mouse IgG-FITC secondary antibody (Santa Cruz Biotechnology, Texas, USA). The nuclei were stained with DAPI (Sigma-Aldrich, St. Louis, MO USA). Images were analyzed using fluorescent microscopy.
Goat anti mouse igg fitc secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Goat anti-mouse IgG-FITC secondary antibody is a laboratory reagent used to detect and visualize the presence of mouse immunoglobulin G (IgG) in biological samples. This antibody is conjugated with the fluorescent dye FITC, which allows for the detection and localization of target proteins through fluorescence microscopy or flow cytometry techniques.
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Lab products found in correlation
Anti cd68 antibody, by Abcam (1 mentions)
Anti mouse cd31 antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Anti non phospho β catenin, by Cell Signaling Technology (1 mentions)
4 protocols using goat anti mouse igg fitc secondary antibody
1
Wound Healing Tissue Collection and Analysis
2
Immunohistochemical Quantification of OPN
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The local protein expression level of OPN in tumor and marginal tissues were assessed using immunohistochemistry. Briefly, the frozen tissue blocks were prepared using Optimal Cutting Temperature (OCT) embedding medium and fixed in 4% paraformaldehyde, and incubated in 20% sucrose. The cryotome was used to prepare 10 nm tissue sections and the slides were washed with PBS and exposed to Triton 3% for 30 min. Triton is used to induce cell membrane permeable to antibodies. In the next step, 10% goat serum was added to the cells for half an hour to block the non-specific antigenic sites. The dilution of 1:100 of a monoclonal antibody of OPN (Santa Cruz Biotechnology, USA, Cat No. # SC-21742) was used for staining in a proper incubation time and the goat anti-mouse IgG-FITC secondary antibody (Santa Cruz Biotechnology, USA, Cat No. # SC-2010) was applied with a delusion of 1:200 in dark at 37 °C. Following adequate washing with PBS (3 times), the 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) (Sigma, Germany,
Cat No. # 28718–90-3) was used to stain the nuclei and then evaluated with a fluorescent microscope. The staining intensity of OPN was quantified using ImageJ and reported as the percentage of positive reactivity[18] (link).
Cat No. # 28718–90-3) was used to stain the nuclei and then evaluated with a fluorescent microscope. The staining intensity of OPN was quantified using ImageJ and reported as the percentage of positive reactivity[18] (link).
3
Immunofluorescence Analysis of DKK3-Mediated β-Catenin Signaling
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The OV-90/PTX and TOV-21G/PTX cells were cultured in a cell culture dish at the density 1 × 104 cells/cm2 and then incubated with DKK3 conditioned medium for 24 h. Subsequently, the cells were fixed with 4% formaldehyde for 15 min and incubated with a permeabilization buffer at 25 °C for 15 min. After blocking with 5% bovine serum albumin, the cells were co-stained with primary FLAG antibody (#ant-146, Prospec, East Brunswick, NJ, USA) and anti-non-phospho-β-catenin (#D13A1, Cell Signaling, Beverly, MA, USA) for 1 h, followed by washing twice with PBS. After incubation with a goat anti-mouse IgG-FITC secondary antibody and a goat anti-rabbit IgG-TR secondary antibody (sc-2010 and sc-2780, respectively, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature, the nucleus was stained using Hoechst 33,442 for 10 min. Fluorescence signals were observed using fluorescence microscopy.
4
Isolation and Characterization of Bat-Derived Virus
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Thirty tissue samples from short-nosed fruit bats were collected from Shaoguan city of China's Guangdong province and homogenized. The homogenate was filtered through a 0.22 μm pore-size filter and used to inoculate confluent monolayers of Vero E6 cells. Blind passages were performed until a cytopathic effect (CPE) was observed. The infected cells were plaque purified, and the virus was propagated in Vero E6 cultures. Virus was collected from infected cells by three freeze-thaw cycles. Aliquots were stored at − 80 °C. One aliquot was titrated on Vero E6 cells to estimate a titer by plaque assay. If CPE was not observed after 4 passages, the result of virus isolation was considered negative. The infected cells were prepared for negative stain and thin section examination by electron microscopy (EM).
In addition, an indirect immunofluorescence assay (IFA) was used to detect MRV proteins in infected cell cultures. Briefly, after washing with PBS, cells were fixed with 4% paraformaldehyde and incubated with 1% BSA for 1 h. Then, the cells were incubated with a mouse anti-MRV (T3D) antibody, followed by a goat anti-mouse IgG-FITC secondary antibody (SANTA CRUZ, USA). After washing, fluorescence was observed under an AMG EVOS F1 inverted microscope. Normal mouse sera, diluted 1:50, was used as a negative control.
In addition, an indirect immunofluorescence assay (IFA) was used to detect MRV proteins in infected cell cultures. Briefly, after washing with PBS, cells were fixed with 4% paraformaldehyde and incubated with 1% BSA for 1 h. Then, the cells were incubated with a mouse anti-MRV (T3D) antibody, followed by a goat anti-mouse IgG-FITC secondary antibody (SANTA CRUZ, USA). After washing, fluorescence was observed under an AMG EVOS F1 inverted microscope. Normal mouse sera, diluted 1:50, was used as a negative control.
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