After the corresponding experimental treatment, 3D4/21 cells were fixed with 4% paraformaldehyde at room temperature for 30 min, treated with 1% Triton X-100 for 10 min, and blocked with bovine serum albumin (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature for 1 h. The samples were then incubated overnight with primary antibody (α-Tubulin antibody, abcam Co., Ltd., Cambridge, MA, USA) at 4 °C. The cells were gently washed with PBST and incubated for 1 h, in the dark, with fluorescently labeled secondary antibodies (abcam Co., Ltd., Cambridge, MA, USA). After gently washing the cells 3 times with PBST, DAPI was added to counterstain cell nuclei. Samples were observed and photographed under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).
Fluorescently labeled secondary antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Fluorescently labeled secondary antibodies are lab equipment designed to detect and visualize target proteins in various applications. They serve as detection reagents, binding to primary antibodies and emitting fluorescent signals that can be measured and analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Confocal laser scanning microscope, by Leica (2 mentions)
Fluorescence microscope, by Leica (1 mentions)
α tubulin antibody, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Axio imager a1 microscope, by Zeiss (1 mentions)
Axiocam mrc, by Zeiss (1 mentions)
700 confocal microscope, by Zeiss (1 mentions)
Blocking buffer, by Thermo Fisher Scientific (1 mentions)
Iba 1, by Abcam (1 mentions)
6 diamidino 2 phenylindole dapi, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Sangon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (1 mentions)
Eif5a1, by Abcam (1 mentions)
Neuronal nuclei (neun), by Abcam (1 mentions)
Mmp 13 antibody, by Abcam (1 mentions)
Collagen 2, by Abcam (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Anti iba1 antibody, by Abcam (1 mentions)
13 protocols using fluorescently labeled secondary antibody
1
Immunocytochemistry of 3D4/21 Cells
2
Immunohistochemical Analysis of Liver Lipid A
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Immunohistochemical analysis was performed as described elsewhere [21 (link)–23 (link)]. For this assay, livers were immediately fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 12 h, then soaked in 30% sucrose at 4°C for 24 h, and stored at -80°C until use. The livers were sliced at 7 or 10 μm thickness on a Microm HM-505 N cryostat. Staining was conducted by means of goat polyclonal antibodies against Lipid A of Enterobacteriaceae bacteria (cat. #: PA1-28903, Invitrogen, USA, 1:200), followed by probing with fluorescently labeled secondary antibodies (Abcam, USA). The slices were treated with the Phalloidin-iFluor 488 Reagent (cat. #: ab176753, Abcam), coverslipped with the Fluoro-shield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; cat. # F6057, Sigma-Aldrich, USA), and visualized under an AxioImager A1 microscope (Zeiss) with camera AxioCam MRc (Zeiss).
3
Histological Assessment of Retinal Infection
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Mice were euthanized, and eyes removed and fixed in 4% paraformaldehyde. Samples were then embedded in paraffin blocks, and 10 µm sections were cut and mounted on slides. Sections were then subjected to de-paraffinization and either H&E staining or blocked overnight at 4°C with blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA). Samples to be evaluated by confocal microscopy were then subjected to an overnight incubation at 4°C with Iba-1 (Abcam, Cambridge, MA, USA) and anti-HSV-1 (Invitrogen, Carlsbad, CA, USA). The tissue was then washed three times and subjected to fluorescently-labeled secondary antibodies (Abcam) overnight at 4°C. Slides were then washed three more times, DAPI was applied, and imaged with a Zeiss 700 confocal microscope (Zeiss, Oberkochen, Germany). Pathological scoring was performed with a masked examiner adapting a previously validated grading system for mouse cytomegalovirus retinitis as detailed in the Table on H&E samples.19 (link)
4
Histological Evaluation of Spinal Cord Injury
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Animals were anesthetized terminally with an overdose of isoflurane inhalation on day 7 after intrathecal transplantation. The spinal cords were embedded in optimal cutting temperature compound. T9–T11 spinal cord segments containing the lesion epicenter were collected for histological evaluation. Briefly, longitudinal sections were deparaffinized with xylene, hydrated in a graded alcohol series, and boiled in citrate buffer (pH 6.0) twice for 5 min each. Subsequently, the sections were cooled and incubated in 3% H2O2 for 15 min at room temperature to inactivate endogenous peroxidases. The slides were then blocked with 10% FBS for 10 min and incubated with rabbit anti-GAP43 or rabbit anti-NF (Abcam) primary antibodies overnight at 4°C. After washing with Tris-buffered saline (TBS), the sections were incubated with fluorescently labeled secondary antibodies (Abcam). Finally, the sections were stained with DAPI and visualized under a confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany).
5
Immunostaining and Confocal Microscopy Protocol
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In total, 5 × 104 cells were seeded into a confocal laser cuvette. After 24 h of cell climbing, 4% paraformaldehyde was fixed for 1 h, 0.25% Triton X-100 was ruptured for 1 h, and 5% bovine serum albumin was blocked for 1 h. Cells were immunostained with antibodies overnight at 4 °C, washed, and incubated with fluorescently labeled secondary antibodies (Abcam, MA, USA) at 37 °C for 1 h, and nuclei were stained with 6-diamidino-2-phenylindole (DAPI) (Beyotime, China). A confocal laser scanning microscope (Leica, Germany) was used for observation and imaging.
6
Histological Analysis of Spinal Cord Lesions
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The tissues from the spinal cord lesions were fixed in 4% paraformaldehyde (Sangon, China), embedded in paraffin, and sliced into 4 μm thick sections. The HE and TUNEL staining were carried out in the presence of HE or TUNEL Staining kits (Yeason, China). For IF staining, sections were blocked with 10% BSA (Beyotime, China) and incubated with anti-NeuN, anti-IBA1, or anti-iNOS antibodies (1:100, Abcam, United States), followed by incubation of fluorescently labeled secondary antibodies (1:250, Abcam, United States) for 1 h at 37°C. The DAPI (Servicebio, China) staining was used to mark the nuclei. The antibody staining was specific.
7
Immunostaining of Rat Brain Sections
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After the rats were anesthetized with isoflurane, the heart was exposed and perfused with pre-cooled PBS to wash away blood in the blood vessels. Perfusion and fixation was then continued using 4% paraformaldehyde. The brain tissue was subsequently dehydrated in 4% paraformaldehyde solution containing graded 10%, 20% and 30% sucrose. The brain was sectioned coronally to the NAc and the sections blocked with 10% goat serum at 37 °C for 30 minutes. Primary antibodies were incubated overnight at 4 °C as follows: eIF5A1 (Abcam, USA, 1:400), Neun (Abcam, USA, 1:500). Sections were then incubated with fluorescently-labeled secondary antibodies (Abcam, USA, 1:1000) at 37 °C for 2 hours. Finally, the nuclei were stained with DAPI and the sections observed by fluorescence microscopy.
8
Immunofluorescence Assay for Collagen II and MMP-13
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Immunofluorescence experiments were carious out according to previously described [49 (link)]. Chondrocytes from various treatment groups were washed twice in PBS before being used to make cell smears. The cells were fixed for 30 minutes at room temperature with 4 percent paraformaldehyde, washed twice with PBS, and then treated for 15 minutes at room temperature with 0.1% Triton-X 100 to render them permeable. After washing the permeate, Collagen II (1:200 dilution, Abcam, UK) and MMP-13 antibody (1:200 dilution, Abcam, UK) were added and the reaction was incubated overnight at 4°C. After washing out the first antibody, a fluorescently labeled secondary antibody (1:200, Abcam, UK) was added and incubated at room temperature for 1 hour before being washed three times with PBS. After adding DAPI staining and washing out the staining solution the cells were observed under the fluorescence microscope (Olympus DP80, Japan).
9
Immunofluorescence Staining of E-cadherin and Vimentin
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Cells were seeded on the coverslips and fixed via 4% paraformaldehyde at 20 °C for 20 min. The attached cells were washed three times using PBS and permeabilized by 0.1% Triton X‐100 for 3 min. The coverslips were blocked by blocking buffer for 1 h, and incubated in presence of primary antibodies against E‐cadherin (1 : 1000; Cell Signaling Technology) and Vimentin (1 : 1000; Cell Signaling Technology) for 2 h. Cells were then treated with a fluorescently labeled secondary antibody (Abcam, London, UK) for 30 min. The cell nuclei were observed by staining with DAPI for 5 min. Images were obtained using a fluorescence microscope.
10
Visualizing Mitochondria and Autophagy
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The cells were added with Mito-Tracker Red CMXRos (1:1000, Invitrogen) 37 °C for 30 min. After washing with PBS for three times, add anti-LC3 antibody overnight at 4 °C. Then add fluorescently labeled secondary antibody (Abcam), for 1 h at room temperature in the dark. The cells were rinsed three times with PBS and observed under fluorescence microscope and photographed.
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