Cryosectioned E15.5 brain sections (20 μm) or slide-mounted adult brain sections (30 μm) were incubated in blocking solution (0.2% Triton X-100, 10% serum, 1% BSA in 0.1 M PBS) for 1 h. Primary antibodies were diluted in 5% serum and 0.1 M PBS and were incubated overnight. Secondary antibodies (1:1,000, Invitrogen) were applied for 1 h at room temperature. After extensive washing, sections were counterstained with DAPI (5 mg ml−1, D3571, Thermo Fisher) for 1 min. ProLong gold mounting media (Thermo Fisher, P36930) were used to secure coverslips. Antibodies used were GFP (1:1,000, ab13970), Tbr1 (1:100, ab31940), Tbr2 (1:100, ab23345), Ctip2 (1:100, ab18465), Satb2 (1:10, ab92446, Abcam), Cux1 (1:50, 13024), and Tuj1 (1:1,000, mms-435p, BioLegend).
P36930
Manufactured by Thermo Fisher Scientific
Sourced in United States
The P36930 is a versatile laboratory instrument designed for a range of scientific applications. It features robust construction and precise temperature control capabilities. The core function of this product is to provide a stable and reliable platform for various experimental and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
A11034, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
A21429, by Thermo Fisher Scientific (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Ab6326, by Abcam (2 mentions)
Ab13970, by Abcam (1 mentions)
D3571, by Thermo Fisher Scientific (1 mentions)
Ab23345, by Abcam (1 mentions)
Ab92446, by Abcam (1 mentions)
Ab31940, by Abcam (1 mentions)
Ab18465, by Abcam (1 mentions)
Mms 435p, by BioLegend (1 mentions)
Volocity imaging software, by PerkinElmer (1 mentions)
Axio observer, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Lsm 880 confocal system, by Zeiss (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Ab9535, by Abcam (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
24 protocols using p36930
1
Immunohistochemistry of Developing Brain
2
Quantitative Retinal Imaging Protocols
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For retinal imaging whole retinas were dissected, flat mounted onto a glass side (RGC layer facing up), placed within an adhesive spacer (Sigma, GBL654006), covered in prolong gold (Thermofisher Scientific, P36930), and coversliped. Images were then taken using a Zeiss LSM 880 confocal system with airyscan imaging mode, followed by airyscan processing using Zeiss Zen software. For mitochondrial volume rendering in optic nerve crush studies, images of RGC axons were acquired at 63x for each sample at a site approximately 100 um from the optic nerve head at axonal fasciculations within the nerve fiber layer (approximately a 10–15 um z-stack). All image analysis was performed using Volocity Imaging Software (Perkin Elmer), mean mitochondrial volume was rendered per image/retina (using the identify objects by intensity module) and consisted of >100 mitochondria. For viral transduction efficiency or RGC density assessment large area imaging studies of whole mounted retinas was conducted. 10x tiled image series were taken with ~60 um stacks and individual RGCs were identified and counted normalized per image area in Volocity or by manual counts in ImageJ, when cells were not easily detected by software. In in vitro hippocampal virus labeling or RGC survival studies, imaging was performed by wide field microscopy using a Zeiss Axio Observer. Cells were counted using ImageJ.
3
Immunofluorescence Imaging of Skin Tissue
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A portion of the dorsal skin was fixed overnight in 10% neutral buffered formalin (NBF) and paraffin embedded. Tissue sections (5 μm) were deparaffinized and rehydrated using graded alcohol concentrations followed by H&E staining or immunofluorescence staining. For IF staining, formalin-fixed and paraffin embedded skin tissue sections were deparaffinized in xylenes and rehydrated using graded alcohol concentrations. Samples were then boiled in 10 mM citric acid for 20 min to retrieve epitopes. Sections were permeabilized with 1% Triton X-100 and blocked with 2% BSA in PBS-T. Primary antibody incubation (anti-myeloperoxidase, 1:100, Abcam ab9535) was carried out overnight at 4 °C in PBS-T + 1% BSA, and secondary antibody incubation (anti-rabbit Alexa-488, 1:500, ThermoFisher A-11034) was performed over 1 h at room temperature. Sections were finally stained for DNA using Hoechst (ThermoFisher, 33342) and mounted (Prolong gold antifade, ThermoFisher P36930). Fluorescence images were acquired on a Zeiss LSM710 confocal microscope and processed using Image J and CorelDraw X5.
4
Myosin 4 Expression in C2C12 Cells
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C2C12 cells were seeded at 1 × 104 cells/cm2 into culture chambers and pretreated with LY294002. After VV stimulation for 3 days and culturing for an additional 6 days (total of 9 days), the cells were washed twice with 1× PBS and then incubated with 4% formaldehyde for 20 min. The cells were then washed 3 times with 1× PBS for 5 min each and then blocked with 5% bovine serum albumin in 1× PBS for 1 h. Subsequently, the cells were incubated with an anti-Myosin 4 (MF-20) primary antibody (1:200; 14-6503-80, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 4 °C overnight. The cells were then washed with 1× PBS containing 0.05% Tween-20 (PBST) for 10 min and then incubated with mouse IgG (H+L) highly cross-adsorbed secondary antibody (A32723, Thermo Fisher Scientific Inc., Waltham, MA, USA). Subsequently, the cells were washed 3 times with PBST for 10 min each, counterstained with Gold Antifade Reagent containing DAPI (1:1000; #8961, Thermo Fisher Scientific Inc., Waltham, MA, USA), mounted with mounting media (P36930, Thermo Fisher Scientific Inc., Waltham, MA, USA) and observed on a Zeiss AxioPlan2 fluorescence microscope (Zeiss Microsystems, Marzhauser, Germany).
5
Quantifying Collagen and Inflammatory Cells in Atherosclerosis
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The Sirius red staining visualized the collagen content, which was analyzed by FIJI (ImageJ). The Sirius red positive area is represented as the percentage of the total atherosclerotic lesion size. Anti-smooth muscle cell antibodies (1A4, 1:1,000, Dako) stained the vascular smooth muscle cells, visualized by the DAB substrate complex. Pro-inflammatory CCR2 expressing macrophages were stained with goat anti-mouse CD107b labeled Alexa Fluor 488 1:100 (Biolegend, M3/84) and rat anti-mouse CCR2 1:100 (Biolegend, SA203G11). Nuclei were visualized by Hoechst 34580 (1:1.000, Sigma, 63493) before mounting them with prolonged gold (Thermo Fischer, P36930). Images were obtained via laser scanning microscopy (LSM700, Zeiss). The counts of CD107b+ positive cells in the atherosclerotic lesions were calculated with FIJI. The CD107b+ and CCR2+ positive cells were counted in the atherosclerotic lesion area with FIJI.
6
Immunohistochemical Analysis of Brain and Liver
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Brain and liver tissue from each treatment group at 9-wk-old and end stage were used for immunohistochemical staining. 24–48 h before sectioning of GFP biodistribution samples, tissues were transferred to a 30% sucrose solution where they remained until sinking. Using a cryostat, brain and liver were sectioned parasagittally at a 30 μm thickness and free-floating sections were collected and washed in PBS containing 0.25% Triton X-100 (PBSt). After a 1-h block at room temperature in PBSt/normal goat serum (NGS, S26-M; Sigma-Aldrich), primary antibodies were diluted in PBSt/NGS and sections incubated overnight. Subsequent to washing in PBSt, appropriate secondary antibodies were diluted in PBSt/NGS and incubated with sections for 30 min at room temperature. Refer to Table S2 for antibodies and dilutions. Filipin complex from Streptomyces filipinensis (F9765; 0.025 g/ml; Sigma-Aldrich) was diluted in PBSt to stain tissue for cholesterol accumulation. ProLong Gold mounting medium with or without DAPI (P36930 or P36935; Life Technologies) was used to coverslip slides after sections were mounted. H&E staining was performed by Histoserv, Inc..
7
Quantifying ADAR2 Nucleocytoplasmic Ratios
8
Immunofluorescence Imaging of MAP2 and ADAR2
9
Co-labeled Extracellular Vesicle Uptake Assay
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Co-labeled Evs were prepared from co-transfection of the plasmids as described above. A total of 2 × 104 cells each of A431 and MCF-7 cells were mixed and seeded to an 8-well chamber slide (Nunc Lab-Tek, Thremo Scientific, Waltham, MA, USA) 24 h before EV treatment. These co-cultured cells were incubated with 2.0 × 107 E626-mCherry or RDG-mCherry co-labeled EVs for 10 min, followed by 3× PBS washes to remove unbound EVs. The cells were fixed with 4% PFA at room temperature for 10 min, washed with PBS three times, and blocked with blocking buffer (1% BSA in PBST) for 60 min. Then, the cells were incubated with the primary antibody (CST D38B1, Cell Signaling Technology, Danvers, MA, USA) in the humidified chamber overnight at 4 °C. After three 5 min PBS washes, they were incubated in the diluted secondary antibody (CST 4412, Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature in the dark. The slide received a coverslip following applying mounting medium (P36930 Life Technologies, Carlsbad, CA, USA) containing DAPI. Fluorescence images were taken at 60× objective magnification by confocal laser scanning microscopy (FluoView FC1000, Olympus, Shinjuku City, Tokyo, Japan).
10
Nile Red Staining of Adipocytes
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Nile red staining was performed following the protocol described by Greenspan et al. with modifications [78 ]. In brief, nile red stock solution of 1 mg/mL concentration was obtained by dissolving 5 mg of nile red powder (Sigma-Aldrich N3013, USA) in 5 mL of acetone. The stock solution was diluted to 1:100 nile red staining solution in 1 mM trizma-maleate (Sigma-Aldrich T3128, USA) and 3% w/v Polyvinylpyrrolidone (Sigma-Aldrich P2307, USA). The differentiated adipocytes were washed with PBS (Sigma-Aldrich D8537, USA) and fixed with 4% paraformaldehyde for 1 h. Then, the cells were stained with nile red to visualize the lipid droplets and DAPI (Life Technologies P36930, USA) to stain the nucleus. Olympus fluorescent microscope was used to observe the stained cells and imaged using cellSens Standard software.
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