To mark PCs, brain sections were incubated with a rabbit anti-calbindin D28K antibody (1:1000, PA1-931, Invitrogen, Waltham, MA) at 4 °C for 24–48 h. After being washed in PBS, sections were incubated with a goat anti-rabbit Cy5 (1:500, A10523, Invitrogen) at room temperature for 3 h. After another rinse with PBS, sections were mounted on slides and cover-slipped with a mounting medium (H-1500, Vector Labs). To identify subtypes of c-Fos positive neurons following the social recognition test, sections were incubated with primary antibodies of guinea pig anti-c-Fos (1:1000, 226308, Synaptic Systems, Gottingen, Germany), rabbit anti-CaMKII (1:200, PA5-99558, Invitrogen) and mouse anti-GAD67 (1:500, MAB5406, Millipore, Burlington, MA) at 4 °C for 24–48 h. After being washed in PBS, sections were incubated with secondary antibodies of goat anti-guinea pig Alexa 488 (1:1000, A11073, Invitrogen), goat anti-rabbit Alexa 555 (1:1000, A21428, Invitrogen) and goat anti-mouse Alexa 647 (1:1000, A21236, Invitrogen) at room temperature for 3 h. After another rinse with PBS, sections were mounted on slides and cover-slipped with a mounting medium (H-1500, Vector Labs). Images were taken with a Zeiss LSM 710 confocal microscope and Zen 2.0 software (Oberkochen, Germany).
H 1500
Manufactured by Vector Laboratories
Sourced in United States, Morocco
The H-1500 is a general-purpose laboratory centrifuge designed for a wide range of applications. It features a compact and durable construction with a maximum speed of 15,000 rpm and a maximum relative centrifugal force (RCF) of 21,500 x g. The H-1500 is suitable for separating and concentrating various biological samples, such as cells, proteins, and nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (7 mentions)
Lsm 700, by Zeiss (4 mentions)
Lsm 780, by Zeiss (4 mentions)
Lsm710 spectral confocal microscope, by Zeiss (3 mentions)
X090930, by Agilent Technologies (3 mentions)
S 1000, by Vector Laboratories (3 mentions)
Dm2500, by Leica (3 mentions)
Ab150080, by Abcam (3 mentions)
W11261, by Thermo Fisher Scientific (3 mentions)
A11008, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Ec plan neofluar 40 1.30 oil dic m27 objective lens, by Zeiss (2 mentions)
Ab150077, by Abcam (2 mentions)
Immunofluorescence microscope, by Olympus (2 mentions)
Ar009, by Boster Bio (2 mentions)
Ti 1000, by Vector Laboratories (2 mentions)
Fv1000 mpe multiphoton laser scanning microscope, by Olympus (2 mentions)
Spc 400, by StressMarq Biosciences (2 mentions)
Autoquant x3, by Media Cybernetics (2 mentions)
S11226, by Thermo Fisher Scientific (2 mentions)
99 protocols using h 1500
1
Immunohistochemical Analysis of Neuronal Subtypes
2
Immunofluorescence Imaging of IL-8 in THP-1 Cells
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THP-1 cells (1 × 106 cells) were washed in PBS and coated on slides using cytospin technique at 600 rpm for 3 min. The slides were then fixed in 4% formaldehyde for 10 min and washed three times in cold PBS. Cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min, washed three times in cold PBS, blocked in 1% bovine serum albumin (BSA) for 1hr, and then incubated overnight with primary antibody (1:100 dilution, rabbit anti-human IL-8 polyclonal antibody; Abcam® ab106350) at room temperature. Cells were washed three times in PBS/Tris buffer and incubated with secondary antibody (goat anti-rabbit antibody conjugated with Alexa Fluor® 488; Abcam® ab150077) for 1hr. After several washes in PBS, the cells nuclei were counterstained with 4′,6-diamidino- 2-phenylindole (DAPI) and mounted (Vectashield, Vectorlab, H1500). Confocal images were collected using inverted Zeiss LSM710 Spectral Confocal Microscope (Carl Zeiss, Gottingen, Germany) and EC Plan-Neofluar 40 ×/1.30 oil DIC M27 objective lens (Carl Zeiss, Gottingen, Germany). After sample excitation using a 405 nm and 488 nm line of an argon ion laser, optimized emission detection bandwidths were configured using Zeiss Zen 2010 software (https://www.zeiss.com/microscopy/int/products/microscope-software.html ).
3
Perfusion, Sectioning, and Imaging Brain Tissue
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Mice were perfused with 4% paraformaldehyde (PFA) and the brain was extracted and fixed for 24 hr at 4°C in 4% PFA, then transferred to 30% sucrose in PBS at 4°C. The brain was mounted on a benchtop microtome and sectioned at 60 µm slice thickness. Free-floating sections were washed in PBS, mounted on glass adhesion slides, stained with DAPI (Vector Laboratories, H-1500), and covered with a glass-slip. In brains used for two-photon imaging we obtained anatomical images in blue for DAPI and in green for GCaMP. In brains used for electrophysiology we obtained anatomical images in blue for DAPI and red for DiI (the electrode had been dipped in DiI before insertion). The LGN border on these images was determined using SHARP-Track (Shamash et al., 2018 (link)).
4
Macrophage and Apoptosis Staining in Aortic Sinus
5
Immunohistochemical and Immunofluorescence Analysis
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Immunohistochemistry (IHC) was performed on formalin-fixed sections and immunofluorescence (IF) on frozen sections. In brief, paraffin-embedded, formalin-fixed sections were deparaffinized and rehydrated. Antigen was recovered by boiling in citrate buffer (H-3300, Vector Laboratories) and incubation in Triton-X, and were digested with proteinase K. Sections were blocked in goat serum and incubated overnight at 4°C for IHC with primary antibodies against β catenin (D13A1, Cell Signaling), F4/80 (ab6640, Abcam), or 4-hydroxynonenal (4-HNE) (ab46545, Abcam). Subsequently, slides were incubated with biotinylated secondary antibody, and the reaction was developed using avidin/streptavidin horseradish peroxidase (PK-4001, Vector Laboratories). For IF, slides were incubated overnight with primary antibody against Ki67 (556003, Becton-Dickinson,) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) (P-0008, Echelon), followed by a secondary antibody labeled with Texas Red (Vector Laboratories) or Alexa Fluor (Invitrogen) and counterstained with DAPI mounting media (H-1500, Vector Laboratories). Dihydroethidium (DHE) stain was performed on frozen liver sections fixed with 4% paraformaldehyde and stained with a 1:1,500 dilution of DHE (10 μg/mL) stain for 10 min at 37°C. Slides were washed twice in PBS and coverslipped. Images were captured using an Olympus BX60 fluorescence microscope.
6
Oocyte Immunofluorescence Staining Protocol
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Fixed oocytes (15 min in 4% PFA; Sigma Aldrich) were permeabilized in 0.1% Triton X-100 for 10 min, washed in PBS supplemented with polyvinyl alcohol (PVA, Sigma Aldrich) and incubated in primary antibodies overnight at 4 °C. The following primary antibodies diluted in PVA/PBS were used: rabbit Ankyrin B (1:150; sc-28560, Santa Cruz), goat Ankyrin B (1:150; sc-14995, Santa Cruz), mouse monoclonal anti-acetylated tubulin (1:150; T6793, Sigma Aldrich), rabbit 4E-BP1(Thr70) (1:500; cs-9455S, CST), rabbit PABP (1:150; sc-28834). Oocytes were then washed 2 × 15 min in PVA/PBS and primary antibodies were detected using relevant Alexa Fluor 488/594/647 conjugates (Invitrogen) diluted to 1:250 for 1 h at room temperature. Washed oocytes (2 × 15 min in PVA/PBS) were mounted onto slides using Vectashield Mounting Medium with DAPI (H-1500, Vector Laboratories). Inverted confocal microscope (Leica SP5) was used for sample visualization. Image quantification and assembly were performed using ImageJ and Adobe Photoshop CS3. Experiments were repeated 3x with 20–30 oocytes per group/experiment.
7
Immunofluorescence Analysis of OX1R and OX2R
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Upon the density of the cell reach to about 70%, we washed them three times with PBS and then fixed the cells with paraformaldehyde for 30 minutes. We chose the goat serum (BOSTER, AR009) to block the samples at room temperature for 30 minutes. After that, we chose the primary antibody against OX1R and OX2R for incubation at 4°C overnight. On the second day, the samples were washed 3 times in PBS. They are incubated with Cy3-labeled secondary antibody (SA00009-2, Proteintech) for one hour at 37°C. Finally, the samples were then counterstained with DAPI (VECTASHIELD, H-1500) for 30 seconds. The images were captured with an immunofluorescence microscope (Olympus, Japan).
8
Immunofluorescence Imaging of NHE3
9
Immunofluorescence Staining of Mouse and Human Cells
10
Fluorescence In Situ Hybridization for Visualizing RNA Foci and MBNL Proteins
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Fluorescence in situ hybridization (FISH) of cultured cells and tissue monolayers was performed as previously described.20 (link) Briefly, cells were fixed and immersed in 70% ethanol at 4°C overnight. After treatment with wash buffer and prehybridization buffer, slides were incubated with (CAG)6CA-5′ Texas red-labeled 2-O-methyl RNA 20-mers probe (IDT). The next day, slides were washed twice and then stained with mounting media with DAPI (H-1500; Vector Labs, Burlingame, CA, USA). For detection of MBNL protein, prior to ethanol treatment, permeabilization with 0.2% Triton 100 in 2× SCC was performed. After probe incubation, cells were incubated with anti-MBNL1 or anti-MBNL2 antibody.
The slides were sealed and imaged at 60× magnification using a Widefield Deltavision microscope. Images were processed by deconvolution with AutoQuant X3 (Media Cybernetics, Rockville, MD, USA) software. Visualization of RNA foci and co-localization of MBNLs proteins were made using ImageJ (National Institutes of Health, Bethesda, MD, USA). For quantification, data were analyzed from at least 10 pictures containing greater than 50 cells each. For cells, data were analyzed from two batches of F35T cells. For tissues, data were analyzed from five FECD tissues.
The slides were sealed and imaged at 60× magnification using a Widefield Deltavision microscope. Images were processed by deconvolution with AutoQuant X3 (Media Cybernetics, Rockville, MD, USA) software. Visualization of RNA foci and co-localization of MBNLs proteins were made using ImageJ (National Institutes of Health, Bethesda, MD, USA). For quantification, data were analyzed from at least 10 pictures containing greater than 50 cells each. For cells, data were analyzed from two batches of F35T cells. For tissues, data were analyzed from five FECD tissues.
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