The olfactory bulbs were xed with 4% paraformaldehyde, embedded in slices, and baked. After the para n sections were completely dewaxed with xylene, 10% calf serum was added, and the section were placed at room temperature for 10 minutes. The sections were incubated with rabbit polyclonal anti-CD11b antibody (1:500, Abcam) at 4 °C overnight, followed by a FITC-labeled goat anti-rabbit IgG (Abcam) at room temperature for 30 minutes. The sections were washed with water, blown dry, sealed with glycerin, and followed by observation under a uorescence microscope (Olympus). TUNEL staining TUNEL staining was carried out according to the manufacturer's instructions (Servicebio). The cells were xed with 4% paraformaldehyde for 30 minutes, ruptured by 0.2% Triton X-100 for 5 minutes, and incubated with 50 µl TUNEL reaction solution at 37 °C for 60 minutes in the dark. DAPI was used to stain the nucleus. The cells were observed and photographed under a uorescence microscope (Olympus). In 6 non-repeating high-power (× 400) elds, the number of TUNEL-positive cells as a percentage of the number of DAPI-positive cells was used to calculate the positivity rate.
Uorescence microscope
Manufactured by Olympus
Sourced in Japan, United States
The Olympus Fluorescence Microscope is an optical instrument designed to detect and analyze fluorescent signals. It utilizes a specialized light source and filter system to excite fluorescent molecules, allowing for the visualization and study of various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (8 mentions)
Rabbit polyclonal anti caveolin 1, by Abcam (6 mentions)
Rabbit polyclonal anti mmp 9, by Abcam (6 mentions)
Rabbit monoclonal anti occludin antibody, by Abcam (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (4 mentions)
Hoechst 33342, by Olympus (4 mentions)
Rabbit monoclonal anti cd31 antibody, by Abcam (4 mentions)
One step tunel apoptosis assay kit, by Beyotime (3 mentions)
Transwell cell culture chamber, by BD (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions)
Paraformaldehyde (pfa), by Solarbio (2 mentions)
Cell light edu apollo567 in vitro kit, by RiboBio (2 mentions)
In nite 200 pro, by Tecan (2 mentions)
Rabbit anti ki67 antibody, by Abcam (2 mentions)
Pi hoechst 33342, by Merck Group (2 mentions)
Hoechst, by Merck Group (2 mentions)
Cck 8, by Beyotime (2 mentions)
Microplate reader, by Molecular Devices (2 mentions)
Cm dil, by Olympus (2 mentions)
134 protocols using uorescence microscope
1
Quantitative Immunofluorescence and TUNEL Assay
2
Fluorescence Imaging of T-2 Toxin Effects
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The nuclei and cytoskeleton of the cells were stained in accordance with the kit's instructions after T-2 toxin (8 nM) treatment for 12 h. Under a uorescence microscope (Olympus, Japan), the uorescence was seen and captured on camera. iFluor™ 488: the uorescence intensity at 517 nm emission wavelength was detected under 493 nm excitation. DAPI: the uorescence intensity at 454 nm emission wavelength was detected under 364 nm excitation. Finally, to evaluate the uorescence intensity, Image-Pro Plus 6.0 was used.
3
Immunohistochemical and Immunofluorescence Analysis of Kidney Tissue
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The formalin-xed kidney tissue was taken for dehydration, transparency, wax dipping, embedding, sectioning, dewaxing, and hydration. Measured the levels of TLR4, p65, and p-p65 antibody.50μl of the corresponding primary antibody was added dropwise at 4℃ overnight. Added the EnVision reagent dropwise and let it stand at room temperature for 30 min. The results were detected by light microscopy with 200×magni cation.Five elds were observed in each sample and the integrated optical density (IOD) was calculated.
Immuno uorescence NRK-52E cells from the different groups were xed with 1 ml of 4% PFA for 25 min, permeated with 0.2% Triton X-100 for 5 min, and blocked with goat serum for 1h. Next, the cells were incubated overnight with primary antibodies against TLR4, followed by incubation with secondary antibodies. The cellular nuclei were stained with DAPI. Cells were sealed with 4μl resistance to uorescence quenching sealing liquid.Images of the stained cells were obtained with a uorescence microscope (Olympus).
Enzyme-Linked Immunosorbent Assay(ELISA)
In ammatory cytokines IL-6 and TNF-α were detected by using conventional ELISA kits(Haling biotechnology Co.,Ltd,Shanghai,China).Operating procedures were strictly nished according to the manufacturer's instructions.
Immuno uorescence NRK-52E cells from the different groups were xed with 1 ml of 4% PFA for 25 min, permeated with 0.2% Triton X-100 for 5 min, and blocked with goat serum for 1h. Next, the cells were incubated overnight with primary antibodies against TLR4, followed by incubation with secondary antibodies. The cellular nuclei were stained with DAPI. Cells were sealed with 4μl resistance to uorescence quenching sealing liquid.Images of the stained cells were obtained with a uorescence microscope (Olympus).
Enzyme-Linked Immunosorbent Assay(ELISA)
In ammatory cytokines IL-6 and TNF-α were detected by using conventional ELISA kits(Haling biotechnology Co.,Ltd,Shanghai,China).Operating procedures were strictly nished according to the manufacturer's instructions.
4
Quantifying Ly-6G+ Cells in MCAO Mouse Brains
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Brain tissues for immuno uorescence analysis were obtained 3 days after MCAO and xed in 4% paraformaldehyde for at least 48 hours. Mouse brains were transferred to a 30% sucrose solution and allowed to dehydrate. Then, 20-mm-thick coronal brain slices were acquired on a cryostat vibratome and incubated with a primary antibody against Ly-6G (1:200, Santa Cruz Biotechnology, CA, USA) at 4°C overnight followed by incubation with a donkey anti-rat antibody conjugated to Alexa 488 (1:200, Jackson ImmunoResearch) for an hour at room temperature. Images were then captured using a uorescence microscope (Olympus, Japan), and the number of Ly-6G-positive cells in the infarction border cortex was counted and analyzed with ImageJ software.
5
Fluorescent Immunodetection of Nephrin and Bax
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All samples were xed with 4% paraformaldehyde for 30 min at room temperature. After being washed three times with PBS, samples were incubated with 0.1% Triton X-100 for 20 min and blocked with goat serum for 30 min. Then kidney sections were incubated with anti-nephrin and anti-Bax (A nity Biosciences, OH, USA), and podocytes were incubated with anti-Bax, anti-p-Akt (A nity Biosciences, OH, USA) at 4℃, overnight. Next, samples were incubated with FITC and Fluor594-conjugated secondary antibodies (A nity Biosciences, OH, USA) for 1 h at room temperature. The nuclei were stained with DAPI (5 μg/mL, Solarbio, Beijing, China) for 10 min and imaged by a uorescence microscope (Olympus, Japan).
6
Quantifying Autophagy and Colony Formation
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Fadu cells were cultured normally for 12 h before the experiment. Cells were incubated in 4% paraformaldehyde (Solarbio, Beijing, China) for 20 min. Cells were washed with PBS and permeabilized with 0.5% Triton X-100 in PBS for 20 min. Next, nonspeci c protein binding sites were blocked with 5%BSA for 2h. Cells were incubated with the following antibodies diluted in PBS with 3%BSA overnight at 4°C: anti-LC3 (1:1000, 3638T, CST, USA). After washing with PBS, cells were further incubated for 1h at 37°C with an anti-rabbit secondary antibody (AF594; Jackson). Samples were next stained with Hoechst 33342 (Aladdin Biochemical Technology Co., Ltd., Shanghai, China) for 10 min. Images were recorded using a uorescence microscope (Olympus, Tokyo, Japan). In 5 randomly selected visual elds at magni cation of 400×.
Colony formation assays 7.5×10 2 cells were plated in DMEM plus 10% Fetal Bovine Serum in 6-well plates and allowed to attach overnight. After 14 days incubation at 37℃ in an atmosphere containing 5% CO 2 . Plates were xed and stained with 0.05% crystal violet. Colonies were counted and data was normalized to the untreated control. Details of cell lines, and incubation times are included in gure captions.
Colony formation assays 7.5×10 2 cells were plated in DMEM plus 10% Fetal Bovine Serum in 6-well plates and allowed to attach overnight. After 14 days incubation at 37℃ in an atmosphere containing 5% CO 2 . Plates were xed and stained with 0.05% crystal violet. Colonies were counted and data was normalized to the untreated control. Details of cell lines, and incubation times are included in gure captions.
7
Immunofluorescence Assay for Myofibroblast Markers
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Primary or immortalized broblasts were seeded on glass slides. After incubation overnight at 37°C, cells were washed with PBS three times, xed with 4% paraformaldehyde at room temperature for 30min, then permeabilized with 0.1% Triton X-100 for 1 h in the 37°C incubator. 10% FBS (Gibco, Cat# 10099141) was added to block the non-speci c antigen. Primary antibodies applied in this assay include myo broblast markers Vimentin (1:500, Abcam, Cat# ab92547, RRID: AB_10562134) and α-SMA (1:500, Abcam, Cat# ab7817, RRID: AB_262054). Goat polyclonal Secondary Antibody to Rabbit IgG (Alexa Fluor ® 594, diluted 1:500) and Goat Anti-Mouse IgG2a (Alexa Fluor ® 488, diluted 1:500) were chosen as secondary antibodies for 1h at room temperature. Counterstained with DAPI and sealed with glycerol, images were taken by a uorescence microscope (Olympus, Tokyo, Japan).
8
Immunofluorescence Analysis of Key Proteins
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In brief, the samples of dewaxed human NPC, nude mice sections and xative-xed CNE1 cells were blocked with ready-to-use 5% bovine serum albumin buffer (Solarbio, Beijing, China) for about 1 hour. After wash with phosphate buffer saline/0.5% tween 20 buffer for at least 5 times, the samples were incubated with dilutive primary antibodies of TP53 (1:100, Bioss, Beijing, China), CASP8 (1:100, Bioss, Beijing, China), MAPK14
(1:100, Bioss, Beijing, China) at 4 °C overnight. Subsequently, the samples were further incubated with corresponding secondary antibody of IgG H&L (Alexa Fluor-488) (1:200, Abcam, USA). Intracellular nuclei were stained using a 4,6-diamidino-2-phenylindole (DAPI), and the targeting positive cells were screened and imaged by a uorescence microscope (Olympus, Japan) before data assays [19] [20] .
(1:100, Bioss, Beijing, China) at 4 °C overnight. Subsequently, the samples were further incubated with corresponding secondary antibody of IgG H&L (Alexa Fluor-488) (1:200, Abcam, USA). Intracellular nuclei were stained using a 4,6-diamidino-2-phenylindole (DAPI), and the targeting positive cells were screened and imaged by a uorescence microscope (Olympus, Japan) before data assays [19] [20] .
9
Cell Proliferation Assay in T24 and 5637 Cells
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T24 and 5637 cells were seeded into 96-well plates at a density of 5´10 3 cells per well. Cell growth was determined using the Cell Counting Kit-8 (CCK8) assay, wherein 10 ml of CCK8 solution was added per well. After incubation for 1 h, absorbance at 450 nm was measured using a microplate reader (In nite 200 PRO, TECAN, Männedorf, Switzerland). All experiments were performed in triplicate. In addition, cell proliferation was also analyzed using a Cell-Light EdU Apollo 567 in vitro kit (C10310-1; Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. Images were visualized and captured under a uorescence microscope (Olympus Corporation).
10
Profilin-1 Immunohistochemistry in Brain Tissue
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Sections of the brain were xed with 4% PFA for 15 min, permeabilized with 0.1% 0.1%Triton®X-100 for 15 min, and after blocked with 10% donkey serum in tris-buffered saline (TBS) for 30 min at RT. The sections were incubated overnight at 4°C with the rabbit anti-PFN1 primary antibody (1:200, Proteintech, China). After rinsing with the TBS, the sections were incubated with the secondary antibody Alexa Fluor®488 donkey anti-rabbit lgG(H+L) for 45 min. Sections were then washed in TBST and counterstained with DAPI for 10 minutes. Finally, the sections were washed with TBST, and 4, 6-diamino-2phenylindole (DAPI, Roche, USA) was used for nuclear staining. The stained tissue sections were then examined with a uorescence microscope (Olympus, Tokyo, Japan), and images were taken at × 200 magni cation.
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