Cryo sections (15 μm thickness) were blocked in 5% donkey serum PBS/Triton X-100 0.25% for 1 hour at RT. Primary antibodies (anti α1 (1:400) (a6f-c, Developmental Studies Hybridoma Bank); anti α3 (1:300) (06172, EMD Millipore, US); anti calbindin (1:400) (ab82812, Abcam, Cambridge, UK) were applied in 1% donkey serum PBS/Triton X-100 0.25% overnight at 4°C. Secondary labelling was done with Alexa Fluor fluorescent-conjugated secondary antibodies (Alexa Fluor 488 donkey anti rabbit (A21206, Life Technologies, Carlsbad, CA, USA); Alexa Fluor 568 donkey anti mouse (A10037, Life Technologies, Carlsbad, CA, USA) (1:350) in 1% donkey serum PBS/Triton X-100 0.25% for 1 hour at RT. Hoechst (1:10000) (Life technologies, Carlsbad, CA, USA) in PBS was used to counterstain the nuclei. Sections were mounted using fluorescence mounting medium (Dako, Glostrup, Denmark) and analysed on a LSM510 laser-scanning confocal microscope using a 40x C-Apochromat water immersion objective NA 1.2 (Carl Zeiss, Göttingen, Germany). Zen 2011 software (Carl Zeiss, Göttingen, Germany) was used for analysis and image capturing.
Ab82812
Manufactured by Abcam
Sourced in United Kingdom, Japan
Ab82812 is a lab equipment product manufactured by Abcam. It is a general-purpose laboratory tool designed for research applications. The core function of this product is to perform a specific task within the laboratory setting, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (4 mentions)
Ab26116, by BioLegend (2 mentions)
Mab354, by Merck Group (2 mentions)
Ab5543, by Merck Group (2 mentions)
Sigma mab4360, by Merck Group (2 mentions)
M3696, by Merck Group (2 mentions)
Sigma ab5062p, by Abcam (2 mentions)
M1406, by Merck Group (2 mentions)
Sigma mab4401, by Merck Group (2 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
A6f c, by Developmental Studies Hybridoma Bank (1 mentions)
Alexa fluor 568 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
C apochromat water immersion objective na 1, by Zeiss (1 mentions)
Zen 2011, by Zeiss (1 mentions)
Mab353, by Merck Group (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Fluoview sv1000 imaging software, by Olympus (1 mentions)
Ab3623, by Merck Group (1 mentions)
9 protocols using ab82812
1
Immunofluorescence Labeling of Cryosections
2
Immunofluorescence Staining of Cell and Brain Samples
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For the immunofluorescence staining, cells and brain sections were blocked with PBS containing 5% normal goat serum (Vector Laboratories), 2% BSA (Gibco) and 0.4% Triton X-100 (Sigma-Aldrich). In the same buffer solution, the cells and sections were then incubated for 24 h with primary antibodies. The following antibodies were used: anti-VEGF (rabbit, 1:500, Invitrogen, ab39250), anti-calbindin (rabbit, 1:500, Chemicon, ab82812 and mouse, 1:500, Abcam, ab9481), anti-S1P (mouse, 1:400, Alfresa Pharma, 274594052), anti-LC-3B (rabbit, 1:200, Cell Signaling Technologies, 3868S), anti-active caspase-3 (rabbit, 1:50, Chemicon, AB3623), anti-β-III tubulin (mouse, 1:400, Chemicon, MAB1637), anti-nestin (mouse, 1:400, Chemicon, MAB353), anti-SSEA-4, TRA-1-60 and TRA-1-81 (mouse, 1:100, Chemicon, MAB4304, MAB4360 and MAB4381). The cells and sections were analysed with a laser scanning confocal microscope equipped with Fluoview SV1000 imaging software (Olympus FV1000) or with an Olympus BX51 microscope. Metamorph software (Molecular Devices) was used to calculate the average intensity.
3
Immunofluorescence Staining Protocols
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Similarly, retinae for immunofluorescence (IHC) were again incubated in 5% PBST and 50% acetone for antigen retrieval and then washed, blocked, and incubated for 3 to 5 days in primary antibody as previously described. Primary antibodies included those against chicken anti-GFP (1:1000, abcam ab290, RRID:AB_303395, Cambridge, UK, abcam.com), rabbit anti-dsRed (1:1000, TakaraBio 632,496, RRID:AB_10013483, Kusatsu, Shiga, Japan, takarabio.com), rabbit anti-Rbpms (1:500, Abcam ab152101), mouse anti-calbindin (1:300, Abcam ab82812, RRID:AB_1658451), rabbit anti-melanopsin (N15, a gift from Ignacio Provencio), and mouse anti-Calretinin (1:300, Sigma Aldrich MAB1568, St. Louis, MO) [43 (link)].
4
Fluorescent Labeling of FABP4 for Kidney Imaging
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Recombinant human FABP4 was labeled with Alexa Fluor 647 reactive dye (AF647, A37573, Thermo Fisher Scientific, MA) according to the manufacturer’s protocol26 (link). Ten minutes after intravenous injection of AF647-FABP4 (15 μg/mouse in 200 μl of saline) or saline alone, kidneys were isolated, longitudinally cut into two equal halves, fixed in Carnoy’s solution (60% ethanol, 30% chloroform and 10% glacial acetic acid) for 6 hours and embedded in paraffin. Immunofluorescence analysis was performed with fluorescein LTL (FL-1321, Vector laboratories, CA) or primary antibodies against THP (sc-19554, Santa Cruz, TX) or CalD (ab82812, abcam, MA). Anti-goat and anti-mouse secondary antibodies conjugated with Alexa Fluor 488 (A-110055 and R37120, respectively, Thermo Fisher Scientific, MA) were used to detect THP and CalD, respectively. Images for immunofluorescent analysis were captured with Biozero-8100 immunofluorescence microscope (Keyence Corporation, Osaka, Japan).
5
Immunohistochemical Analysis of Apoptosis and Cellular Markers
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Immunohistochemistry was performed principally by the following methods previously described27 (link). Sections were pre-treated with Proteinase K before TUNEL staining using ApopTag peroxidase in situ apoptosis detection kit (Millipore). The staining was amplified using biotin-conjugated secondary antibodies, VECTASTAIN ABC system (Vector) and TSA Plus system (Perkin Elmer). Monoclonal anti-BrdU (1:100, Clone 3D4, BD Biosciences, 555627), anti-Calbindin (1:1000, Abcam, ab82812), anti-Tuj1 (1:1,000, Abcam, ab78078), anti-Nestin (1:50, rat-401, DSHB), anti-FLAG M2 (1:50, Sigma, F1804) and anti-NeuN (1:500, Clone A60, Millipore, MAB377) antibodies were used. Polyclonal anti-HSF1X50 (link) (1:500), anti-HSF1 antibody (1:200, C-19, SCBT, sc-8061) and anti-Cutl1 (1:100, M-222, SCBT, sc-13024) antibodies were also used. The HSF1 expression pattern in Supplementary Fig. 5 was consistently obtained by using both HSF1X and HSF1 C-19 antibodies.
6
Immunofluorescent Analysis of Inhibitory Neurons
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Inhibitory human neurons were fixed for 15 minutes in 4% paraformaldehyde in phosphate buffered saline (PBS) and permeabilized using 0.1% Triton X-100 in PBS for 10 minutes at room temperature. Cells were then incubated in blocking buffer (4% bovine serum albumin (BSA) with 1% normal goat serum in PBS) for 1 hour at room temperature and then incubated with primary antibodies diluted in blocking buffer for 1 hour at room temperature, washed with PBS three times, and subsequently incubated in secondary antibodies for 1 hour at room temperature. Confocal imaging analysis was performed using a Zeiss LSM700. Primary Antibodies used include: mouse anti Oct4 (Millipore Sigma MAB4401, 1:2000), mouse anti Tra-1-60 (Millipore Sigma MAB4360, 1:1000), mouse anti MAP2 (Sigma-Aldrich M1406, 1:500), rabbit anti MAP2 (Sigma-Aldrich M3696, 1:500), chicken anti MAP2 (Millipore AB5543, 1:1000), rabbit anti Synapsin (e028, 1:3000), rabbit anti VGAT (Millipore Sigma AB5062P, 1:2000), mouse anti Gad-67 (Abcam ab26116, 1:500), mouse anti β3 Tubulin (BioLegend 801201, 1:2000), mouse anti Calbindin (Abcam ab82812, 1:500), guinea pig anti Parvalbumin (Swant GP72, 1:2500), rat anti Somatostatin (Millipore MAB354, 1:50).
7
Fluorescence Immunostaining of Renal Tissue
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Fluorescence immunostaining was performed in formalin-fixed, paraffin-embedded tissue sections, using FPN antibody (Novus Biologicals; NBP1-21502) at 1:100 and Pro-Hepcidin(AA 39-59) antibody at 1:50 (Antibodies Online; ABIN350367). The specificities of the FPN and hepcidin antibodies were confirmed using Fpnfl/fl, Pax8.CreERT2+ animals and Hamp–/– animals as negative controls, respectively (Supplementary Figure S1 ). Renal segment markers were identified using aquaporin-1 antibody at 1:200 (Biotechne; NB600-749), aquaporin-2 antibody at 1:200 (Biotechne; NBP1-70378), or calbindin antibody at 1:100 (Abcam; ab82812). Secondary antibodies were anti-rabbit IgG Alexa Fluor-488 (Abcam; ab150073), anti-donkey IgG Cy3 (Abcam; ab6949), and anti-mouse IgG Alexa568 (Abcam; ab175473). Slides were imaged using an FV1000 Olympus microscope.
8
Immunofluorescence Labeling of Neural Cells
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Antibodies: goat anti-GFAP (ab53554, 1:300), rabbit anti-Calbindin (ab11426, 1:1000), mouse anti-Calbindin (ab82812, 1:1000), donkey anti-rabbit (ab175649, 1: 100) all from Abcam and donkey anti-goat (A11057, 1:500), Vybrant CM-DiI cell labelling solution (V-2288) from Life Technologies, mouse anti-NeuN clone A60 (MAB377, 1 in 100), mouse anti-GFAP (MAB360, 1:500), rabbit anti-collagen type IV (AB756P, 1:500), anti-nuclei clone 3E1.3(MAB4383) from Millipore, rabbit anti-cleaved caspase 3 (5A1E, 1:500) from Cell Signaling Technology, Click-iT EdU Alexa Fluor-647 imaging kit from Molecular Probes. HGF and EGF were ordered from Peprotech and used at a final concentration of 50 ng/mL and 30 ng/mL respectively. All the OCSC culture reagents have been described in43 (link).
9
Immunofluorescent Analysis of Inhibitory Neurons
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