Islets were harvested, fixed in 4% paraformaldehyde, and immersed in 30% sucrose solution to be embedded in a frozen block. Inflammatory markers and dedifferentiation markers were detected in cell sections by immunofluorescence. Anti-FoxO1 (Santa Cruz, Dallas, Texas, USA), anti-cleaved caspase 3 (Cell Signaling, Danvers, MA, USA), anti-Oct4 (Abcam, Cambridge, UK), anti-NGN3 (Abcam), anti-A11 oligomer (Invitrogen), anti-Pdx1 (Abcam), anti-IL-1β (Abcam), and anti-insulin (Abcam) were used as primary antibodies. M1/M2 macrophage markers were identified using the antibodies of M2 macrophage differentiation markers (anti-arginase-1; Cell Signaling) and M1 macrophage differentiation markers (CD80; Abcam). Sections were incubated with primary antibody diluted in blocking buffer overnight at 4°C. Secondary antibodies (Alexa Fluor® 488-conjugated goat anti-rabbit, Alexa Fluor® 488-conjugated goat anti-mouse, and Alexa Fluor® 594-conjugated goat anti-rabbit; Abcam) were added and incubated for 1 h at room temperature. Counterstaining was performed using DAPI (1 : 10,000). Images were obtained from each section using a fluorescent microscope (Olympus, Shinjuku, Tokyo, Japan).
Alexa fluor 488 conjugated goat anti mouse
Manufactured by Abcam
Sourced in United States
Alexa Fluor-488 conjugated goat anti-mouse is a fluorescent-labeled secondary antibody. It is designed to detect and visualize mouse primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated goat anti rabbit, by Abcam (2 mentions)
Anti anp antibody, by R&D Systems (2 mentions)
Alexa fluor 647 conjugated donkey anti goat, by Abcam (2 mentions)
Anti oct4, by Abcam (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Anti pdx1, by Abcam (1 mentions)
Anti insulin, by Abcam (1 mentions)
Anti ngn3, by Abcam (1 mentions)
Anti arginase 1, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit, by Abcam (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Anti foxo1, by Santa Cruz Biotechnology (1 mentions)
Facscanto 2, by BD (1 mentions)
Dmi6000, by Leica (1 mentions)
Alexa fluor 647 conjugated goat anti chicken, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole dapi solution, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti cox 2, by Abcam (1 mentions)
Anti nlrp3, by Novus Biologicals (1 mentions)
6 protocols using alexa fluor 488 conjugated goat anti mouse
1
Islet Inflammatory and Dedifferentiation Analysis
2
Evaluating Cell Surface Antigens by FACS
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Cell surface antigens on cells were evaluated by fluorescence activated cell sorting (FACS) analysis. For FACS, cells were dissociated by 0.05% trypsin/EDTA (Highclone), washed with PBS, fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and blocked with a mixture of bovine serum albumin (BSA). Next, samples were stained with antibodies against Anti-Myh6 Actin (alpha myosin heavy chain actin, Abcam, 1:200) and Anti-ANP antibody (R&D, 1:200) for 30 min or 1 h at 4 °C. Also, Anti-Goat IgG was used for isotype control. Samples were subsequently stained with fluorescently labeled secondary antibodies (Alexa Fluor-488 conjugated goat anti-mouse (Abcam, 1:400) and Alexa Fluor-647 conjugated donkey anti-goat (Abcam, 1:400)) for 30 min at 4 °C. Corresponding mouse/rabbit isotype antibodies were used as controls. Cell immunotypes were determined by BD FACS CantoII (BD Biosciences) and the percentage of expressed cell surface antigen was calculated for 10,000 gated-cell events.
3
Immunohistochemical Visualization of Cardiac Markers
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Samples were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and blocked with a mixture of BSA. Samples were incubated with primary antibodies (Anti-Myh6 Actin (Abcam, 1:200) and Anti-ANP antibody (R&D, 1:200) overnight at 4 °C and washed. Samples were subsequently stained with fluorescently labeled secondary antibodies (Alexa Fluor-488 conjugated goat anti-mouse (Abcam, 1:400), Alexa Fluor-647 conjugated donkey anti-goat (Abcam, 1:400)) for 30 min at at RT and 4′,6-diamidino- 2-phenylindole dihydrochloride (DAPI) as a nuclear counterstain for 5 min at R.T. Samples were washed and mounted for imaging on a fluorescence microscope (Leica DMI-6000, Leica). The relative surface area of coverage for stains was quantified with ImageJ software (NIH).
4
Immunohistochemical Analysis of tMCAo-Induced Changes
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For IF AT8 (1:500) and MBP (1:2000 polyclonal antibody, Invitrogen) were incubated overnight, followed by the secondary antibodies that included Alexa Fluor® 488 conjugated Goat anti-mouse (1:1000) and Alexa Fluor 647-conjugated Goat anti-chicken (1:200; both from Abcam). Sections were mounted on glass slides with 4′,6-diamidino-2-phenylindole (DAPI) solution (Vectashield, from Vector laboratories). The omission of the primary antibodies resulted in no staining. The sections were photographed at 40× magnification and the images were used to evaluate the fluorescence intensity of MBP immunostaining in regions of interest located in the corpus callosum and stratum alveus. Images were obtained using the laser Olympus FV 1000 D microscope and IF was measure with Fluoview FV1000 software. P-tau was evaluated by calculating the total number cells immuno-stained with a signal larger than 8 microns. All analyses were performed per mouse and for each mouse three slides were measured (Nguyen and Nguyen, 2013 ).
As tMCAo can cause changes in the posterior circulation as well (Lee et al., 2014 (link)), we examined the hippocampal formation and the ipsilateral hemisphere.
As tMCAo can cause changes in the posterior circulation as well (Lee et al., 2014 (link)), we examined the hippocampal formation and the ipsilateral hemisphere.
5
Immunofluorescent Analysis of Cardiac Tissue
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Hearts were resected and fixed in 4% paraformaldehyde for at least 1 day to prepare for immunofluorescence analysis. Thereafter, the specimens were soaked in gradient sucrose solutions in PBS to dehydrate and were processed into frozen sections. Representative sections from the ischemic region were permeabilized in 0.5% Triton X-100 for 15 min and sealed with 5% BSA for 1 h. Afterwards, the sections were incubated with the following primary antibodies at 4°C for 12 h: anti-COX2 (1:100, Cat no. ab15191; Abcam), anti-NLRP3 (1:100, Cat no. NBP2-12446; Novus, Littleton, CO, United States), anti-Vimentin (1:100, Cat no. 5741; CST) and anti-Smooth Muscle Actin (1:100, Cat no. sc-53142; Santa Cruz Biotechnology, Santa Cruz, CA, United States). Primary antibodies were detected by Alexa Fluor 488-conjugated goat anti-rabbit (1:500, Cat no. ab150077; Abcam), Alexa Fluor 568-conjugated goat anti-rabbit (1:500, Cat no. ab175471; Abcam), or Alexa Fluor 488-conjugated goat anti-mouse (1:500, Cat no. ab150113; Abcam) secondary antibodies, respectively, for 2 h at room temperature. Nuclei were identified with its marker of 4′,6-diamidino-2-phenylindole (DAPI) (Cat no. 62248; Thermo Fisher Scientific). Finally, the sections were observed with a fluorescence microscope.
6
Brain Tissue Immunofluorescent Staining
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After the mice were perfused, brain tissue samples were fixed in phosphate buffered 4% paraformaldehyde at 4°C overnight. The mouse brain was then cut into 5-μm thick coronal sections for immunofluorescent staining. Slices were incubated with primary antibodies including mouse anti-ZO-1 monoclonal antibody (1:300, Abcam, Cambridge, MA, United States) and mouse anti-Occludin monoclonal antibody (1:100, Abcam, Cambridge, MA, United States) overnight at 4°C. Then, the sections were washed 3 times with PBS (pH = 7.4), and were incubated with a secondary antibody (Alexa Fluor 488 conjugated goat anti-mouse, Abcam, Cambridge, MA, United States) for 1 h at room temperature. Finally, sections were washed with PBS and stained with DAPI. All sections were visualized and photographed by using a fluorescence microscope (Olympus Co., Japan).
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