For double immunofluorescent staining, the same sections for immunostaining were prepared and stained as follows. First, we stained nuclei using 4′,6-diamidino-2-phenylindole dihydrochloride. After that, the sections were rinsed well with PBS and incubated with primary antibody (anti-CD11c antibody [1:400 dilution, No. 17342-1-AP], anti-TSLPR antibody [goat anti-mouse TSLPR polyclonal antibody, AF546, R&D Systems, Minneapolis, MN, USA], or anticytokeratin antibody (undiluted solution of monoclonal mouse anti-human cytokeratin clone AE1/AE3 ready-to-use, IS053, DAKO) at 4°C overnight. Third, the sections were incubated with secondary antibody (1:500 dilution of donkey anti-goat IgG, Alexa Fluor 488 [A32814, Thermo Fisher Scientific, Tokyo, Japan], 1:500 dilution of donkey anti-rabbit IgG, Alexa Fluor 647 [A32733, Thermo Fisher Scientific], or 1:500 dilution of donkey anti-mouse IgG, Alexa Fluor 594 [A32744, Thermo Fisher Scientific]) at room temperature for 60 min, and the coverslip was mounted. Images of the sections were taken using a fluorescence microscope (Confocal Quantitative Image Cytometer, CQ1 Yokogawa, Tokyo, Japan).
A32744
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The A32744 is a high-performance laboratory equipment used for precise analytical measurements. It is designed to provide accurate and reliable results for a wide range of applications. The core function of this product is to perform sensitive and precise measurements, though its specific intended use may vary depending on the customer's needs and requirements.
Automatically generated - may contain errors
Lab products found in correlation
Mab377, by Merck Group (3 mentions)
A32814, by Thermo Fisher Scientific (2 mentions)
Ab5076, by Abcam (2 mentions)
A32731, by Thermo Fisher Scientific (2 mentions)
A32790, by Thermo Fisher Scientific (2 mentions)
A32766, by Thermo Fisher Scientific (2 mentions)
A32754, by Thermo Fisher Scientific (2 mentions)
A32733, by Thermo Fisher Scientific (1 mentions)
Ab6721, by Abcam (1 mentions)
Alexa fluor 594 conjugated donkey anti goat, by Thermo Fisher Scientific (1 mentions)
A11058, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Gtx85454, by GeneTex (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
P36930, by Nikon (1 mentions)
Sc 32245, by Santa Cruz Biotechnology (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Hm525 nx, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
10 protocols using a32744
1
Double Immunofluorescent Staining Protocol
2
Immunohistochemical Analysis of Brain Markers
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After brain sections were deparaffinized, endogenous peroxidase activity was blocked by immersion in hydrogen peroxide for 10 min. Then, the sections were incubated with 5% normal donkey serum for 1 h to block non-specific binding. Subsequently, the sections were incubated at 4°C overnight with the following primary antibodies as appropriate (Table S1): rabbit anti-PYGB (HPA031067, 1:50, Atlas), rabbit anti-GYS1 (1:25, Abcam), mouse anti-NeuN (MAB377, 1:200, Millipore), chicken anti-GFAP (GTX85454, 1:500, GeneTex), and goat anti-Iba1 (ab5076, 1:300, Abcam). The sections were then treated with fluorescent secondary antibodies as follows: AlexaFluor 488 (green)-conjugated goat anti-rabbit (A32731, 1:500), AlexaFluor 594 (red)-conjugated goat anti-chicken (A32759, 1:500), AlexaFluor 594-conjugated donkey anti-mouse (A32744, 1:500), and AlexaFluor 594-conjugated donkey anti-goat (A11058, 1:500) (all from Thermo Fisher Scientific). Nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole; 1:1000; Sigma-Aldrich, St Louis, MO, USA). Biotinylated secondary antibody (ab6721, 1:5000, Abcam) and peroxidase-conjugated streptavidin followed by chromogenic reaction with 3,3’-diaminobenzidine were added to sections for immunohistochemical staining, which were counterstained with hematoxylin.
3
Immunohistochemical Analysis of Annexin A6 Expression in Mouse Brain
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Two- to four-month-old heterozygous annexin A6GFP or wild-type mice were euthanized and transcardially perfused with ice-cold PBS containing protease and phosphatase inhibitors. After perfusion, the brain was bisected, and 1 hemibrain was drop-fixed in 4% paraformaldehyde/PBS and cryopreserved in 30% w/v sucrose/PBS for sectioning. The other hemibrain was flash-frozen in liquid nitrogen for biochemical analysis. A total of 30 μm coronal floating brain sections were cut and stained as follows. Sections were washed 3 times in TBS, incubated in 16 mM glycine in TBS-T, and blocked first in 5% donkey serum in TBS-T, then in 1% BSA in TBS-T. Sections were incubated overnight at 4°C with anti-TurboGFP (1:500) and mouse anti-NeuN (MilliporeSigma, MAB377, 1:1000) in 1% BSA TBS-T. The following day, they were incubated with 1:750 donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific catalog A32790) and donkey anti-mouse Alexa Fluor 594 ( Thermo Fisher Scientific catalog A32744). All staining was performed at the same time. Sections were mounted with ProLong Gold Antifade (Thermo Fisher Scientific catalog P36930) and images acquired on a Nikon A1R or W1 confocal microscope with a 20× or 40× objective, using NIS-Elements software. All image acquisition settings were the same among genotypes.
4
Immunostaining of Muscle Tissue and Cells
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Serial cross-sections (10 μm thick) from each frozen left GC muscle were transversally cut on a cryostat microtome set at −20°C (HM 525 NX, Thermo Fisher Scientific) and mounted on slides (SuperFrost® Plus, Menzel Gläser, Thermo Fisher Scientific). 2B4 and SF1 cells were grown on plates at seeding density of 2.5×104 for 11 days. Tissue sections and cells were fixed in paraformaldehyde for 15 min at room temperature (RT) and permeabilized with 0.1% TWEEN 20 (Sigma-Aldrich) and 0.1% bovine serum albumin (Sigma-Aldrich) in PBS for 45 min at RT. After three washes with PBS, sections and cells were blocked in saturation buffer (0.1% bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer. After three washes in PBS, sections and cells were incubated with 488/594 Alexa Fluor-conjugated secondary antibodies (1:1000; A21206 and A32744, Thermo Fisher Scientific) for 45 min at RT. Nuclei were stained with PureBluTM Hoechst 33342 Nuclear Staining Dye (1:50; Bio-Rad). Images were captured using a dark-field microscope (CL-I Eclipse Nikon) at 20× magnification.
5
Immunofluorescence Analysis of EMT Markers
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After overnight culture, cells were seeded on slides and adherent. Slides were treated with 4% formaldehyde, incubated with 0.1% Triton/PBS three times to permeabilize the cells, cleaned with PBS, and blocked with 0.1% Tween‐100 in 2% BSA for 1 h. Then the slides were incubated with primary N‐cadherin and vimentin antibodies (1:500) at 4°C for 16 h, followed by fluorescent antibodies (1:200, Invitrogen, A32744, A32740) for 40 min and stained with 4′,6‐diamidino‐2‐phenylindole (DAPI; ab104139, Abcam). Random fields were chosen to capture images with fluorescent microscopy (DMi8, Leica).
6
Immunostaining of Embryonic Gonad Sections
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Embryonic gonads were fixed in 4% PFA for 30 min at 4°C. Gonads were next submerged in 10 and 20% sucrose in PBS for 1 h each at 4°C, and in 30% sucrose in PBS overnight at 4°C. The gonads were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetek) and frozen in liquid nitrogen. Six-micrometer-thick sections of each gonad were applied to glass slides and autoclaved in TRS (Dako). After pre-incubation with 3% skim milk in PBS-T (PBS with 0.1% Tween20 (Sigma-Aldrich)) at RT for 30 min, the sections were reacted with primary antibodies overnight at 4°C at the following dilutions: 1:200 for mouse anti-phospho-histone H3 (ser10) (1:200, Sigma-Aldrich, 06-570), goat anti-CDH1 (1μg/ml, R&D, AF748), rabbit anti-DNMT3L (1:500, provided by Dr. Yamanaka), rabbit anti-STRA8 (1:200, abcam #ab49602), rabbit anti-Ki67 (1:200, Invitrogen #MA5-14520), rat anti-Ki67 (1:100, Invitrogen AB_10854564) and anti-pS6 (1:200, CST #2211). Secondary antibodies labeled with Alexa 488, 594 or 647 (1:1,000, Invitrogen A21207, A21208, A21209, A32766, A32814, A32754, A32744 and A32795) were used. DNA was counter-stained with DAPI (100 ng/ml). Fluorescence microscopy was performed using Olympus FV1200 and images were processed with ImageJ/Fiji (Schindelin et al., 2012 (link)).
7
Immunofluorescent Labeling of Brainstem and PVT
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Sections of the brainstem that contained CTB labeled neurons or mCherry neurons were incubated in a solution containing a mouse anti-TH monoclonal antibody (1:10,000; T-2928, Sigma-Aldrich, Oakville, ON, Canada) for 2 days. After several rinses, sections were transferred to a secondary antibody solution containing Alexa-Fluor Plus 647 (1:3000, A32787, Invitrogen) for CTB sections and Alexa-Fluor Plus 488 (1:3000, A32766, Invitrogen) for mCherry sections for 2 h. Sections of PVT were incubated in the same TH antibody solutions before transferred to a secondary Alexa-Fluor Plus 594 donkey anti-mouse antibody (1:3000; A32744, Invitrogen) for 2 h.
8
Immunohistochemical Analysis of Neuronal Markers
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Mice were anesthetized with sevoflurane, and brains were cut on a sliding freezing stage microtome in 30 μm thick sections. Hippocampal sections were washed 3 times in PBS. Following 15 min permeabilization with 0.3% Triton X-100 in 0.01 M PBS, sections were blocked with 10% donkey serum for 2 h. Next, sections were incubated overnight at 4°C with the following primary antibodies: DHX9 (1:300, 17721-1-AP, Proteintech), NeuN (1:200, ab104224, Abcam), Iba1 (1:200, ab5076, Abcam), GFAP (1:200, ab3554, Abcam), MAP2 (1:200, ab11267, Abcam), FMRP (1:100, sc-101048, Santa Cruz), STAU1 (1:100, sc-390820, Santa Cruz), or c-Fos (1:200, ab208942, Abcam). The next day, slides were washed 3 times for 7 min in PBS. Then, sections were incubated with the corresponding fluorescent-conjugated secondary antibody (A32766, A32744, A32790, A32754, Invitrogen) for 2 h at room temperature in the dark. DAPI Fluoromount-G aqueous mounting medium with anti-fade properties (SouthernBiotech) was used to identify cell nuclei. Slides were stored in the dark at 4°C until needed for analysis. Fluorescence images were captured with an Olympus FV1000 laser confocal microscope. For c-Fos positive neuron analysis, ImageJ was used to analysis the signals of hippocampal neurons from 3 animals per condition.
9
Comprehensive Immunofluorescence Imaging Protocol
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Cells were plated on coverslips and fixed in 4% paraformaldehyde for 30 min. Then, the membranes were permeabilized using 0.2% Triton X-100. The cells were blocked with 5% BSA solution for 1 hr at room temperature. Cells were incubated with specific primary antibodies overnight at 4°C. Caspase 3 (CST #9662S), α-tubulin (CST, #3873), acetylated tubulin (Proteintech, USA, #662001-IG), γ-tubulin (Proteintech, #15176-1-AP), pericentrin (Abcam, ab4448), CRB3 (Sigma, USA, HPA013835), EEA1 (BD, USA, #610456), Rab11 (BD, #610823), and GCP6 (Abcam, ab95172) were used as primary antibodies. The secondary antibodies were Alexa Fluor 488-labeled or 594-labeled (Invitrogen, A32731, A32744). DPIA (5 μg/ml) was used for DNA staining, and images were captured using a confocal microscope (Leica SP5 II).
10
Immunohistochemical Analysis of Brain Tissue
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Four weeks post-injection, animals were transcardially perfused with 1× phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA). Brains were extracted and subsequently fixed in 4% PFA overnight at 4 °C. Brains were then immersed in 30% sucrose (prepared in 1× PBS), at 4 °C, until equilibrated in sucrose mixture. Brains were embedded in a 1:2 OCT (Tissue Tek, Torrance, CA) and 30% sucrose mixture, and cryo-sectioned at 40 μm (Cryostar NX70, ThermoScientific, Waltham, MA). Sections were permeabilized in 0.5% TritonX-100 for 1 h, blocked in 5% goat serum (10% normal goat serum, 50062Z, Life Technologies) for 1 h, and then incubated in primary antibody (anti-NEUN, 1:1000, EMD Millipore MAB377; anti-GFAP, 1:500, EMD Millipore MAB360; anti-OLIG2, 1:200, Abcam ab109186; anti-IBA1, 1:1000, Wako Chemicals NC9288364; anti-GFP, 1:800, Invitrogen A11122) overnight at 4 °C. Sections were washed three times in 1× PBS and incubated in secondary antibody (anti-mouse, Invitrogen A32744; or anti-rabbit, Invitrogen A32740) for 1 h at room temperature. Sections were washed three times in 1× PBS and mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA).
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