For puncta formation, cells were plated on coverslips and transfected with 4 µg/ml of EGFP-LC3 plasmid (Addgene, 11546) 24 h prior to CBR and/or CQ treatment. After 24 h, cells were fixed using 4% paraformaldehyde and mounted using mounting medium (Dako, S3023). For β3-tubulin immunofluorescence, cells were plated on coverslips and after 24 h, cells were fixed using 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in PBS and blocked in 1% bovine serum albumin (BSA) for 30 min at room temperature. After rinsing, the cells were incubated with a mouse anti-β3-tubulin antibody (Santa Cruz, sc-80005), diluted 1:250 in 2% BSA. After overnight incubation at 4 °C, the cells were washed with PBS and incubated in goat anti-mouse conjugated to Alexa Fluor 488 (Invitrogen, A-11001), diluted 1:2000 in 2% BSA. After rinsing, cells were stained for an additional 30 min with TO-PRO3 (Life Technologies, T3605), diluted at 1:1000 in PBS. Cells were then washed and mounted using mounting medium (Dako, S3023). Imaging was performed using Zeiss LSM 510.
S3023
Manufactured by Agilent Technologies
Sourced in United States, Germany, Denmark, Japan
The S3023 is a laboratory instrument designed for analytical applications. It is capable of performing precise measurements and analysis of samples. The core function of the S3023 is to provide accurate and reliable data to support scientific research and testing.
Automatically generated - may contain errors
Lab products found in correlation
D9542, by Merck Group (14 mentions)
Lsm 700, by Zeiss (13 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (11 mentions)
Axio observer z1, by Zeiss (9 mentions)
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Lsm 510, by Zeiss (6 mentions)
Lsm 780, by Zeiss (6 mentions)
Lsm 880, by Zeiss (6 mentions)
Lsm 780 confocal microscope, by Zeiss (6 mentions)
P6148, by Merck Group (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Zen software, by Zeiss (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
Sytox green, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Cm1950, by Leica (2 mentions)
163 protocols using s3023
1
Quantifying Autophagosome Formation and Neuronal Cytoskeleton
2
Immunofluorescence Staining of Tumor Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Histological analyses were done on snap frozen tissue harvested from tumor bearing mice. Briefy, sections were fixed with acetone for 10 min and non-specific antigens were blocked in PBS containing 2% FCS for 15 min. Staining for CCL5/RANTES was done using anti-RANTES (ab 9679; lot GR5419-63); VL-4 to stain LCMV or anti-NK1.1 (Cat 4322177 eBioscience). Antibodies were diluted 1:100. After 1 h of incubation, sections were washed with PBS containing 2% FCS and incubated for 1 h with secondary antibodies (dilution of 1:100). After mounting (S3023, Dako), images were acquired with a fluorescence microscope (KEYENCE BZ II analyser). Immunofluorescence was performed using cells grown on coverslips. Different human melanoma cells (Ma-Mel-86a, Ma-Mel-51 and Ma-Mel-86c) were grown (2 × 105 cells per well in a 24 well plate) on cover slips. After fixation with 4% Formalin for 30 min, Triton X solution (1%) was added and incubated for 20 min room temperature for permeabilization. After washing, 10% FCS in PBS was added per well to block non-specific binding followed by a 1 h incubation. Primary antibodies were added and samples were incubated for 1 h at room temperature. After washing, secondary antibodies were added (1:100 dilution) followed by a 1 h incubation, washing and mounting (S3023, Dako). Images were acquired with a fluorescence microscope (KEYENCE BZ II analyser).
3
Quantifying Adult-Born Hippocampal Neurons in Nr2e1 Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the survival of adult-born hippocampal neurons in Nr2e1−/− mice, sections were double-labelled with BrdU and the neuronal marker NeuN. Sections were washed, DNA strands denatured by incubation in 2 M HCl for 45 min at 37°C, renatured in 0.1 M sodium tetraborate (pH 8.5) and then blocked in 3% normal donkey serum (NDS; Sigma D9663) to prevent non-specific binding. Sections were incubated with anti-BrdU antibody (Abcam; AB6326; 1:250) followed by AlexaFluor594 donkey anti-rat (Abcam; AB150156; 1:500) and NeuN (Millipore; MAB377; 1:100) antibodies. Sections were then incubated with AlexaFluor488 donkey anti-mouse antibody (Abcam; AB150105; 1:500), washed and coverslipped using anti-fade medium (DAKO; S3023). The number of microglia in the hippocampi of these animals was assessed by staining for ionised calcium binding adaptor molecule 1 (Iba-1). Sections were washed, incubated in 3% NDS and then in anti-Iba-1 antibody (Wako; 019-19741; 1:1000) overnight. Sections were then incubated in AlexaFluor488 donkey anti-rabbit antibody, counterstained with DAPI (Sigma; D9642; 1:5000) and coverslipped with anti-fade mounting medium (DAKO; S3023).
4
Immunofluorescence Staining for Cardiomyocyte Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescence staining, cells were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich, 158127) for 10 min at room temperature (RT) and washed three times with DPBS without Ca2+/Mg2+. Cells were then permeabilized with 0.25% Triton X-100 (Sigma-Aldrich, X100) for 15 min at RT before washing three times with DPBS, followed by incubation with 10% FBS in DPBS containing 0.1% TritonX-100 (PBST) for 1 h at RT. Primary antibodies against cardiac Troponin (cTNT), α-actinin, MLC2v, MLC2a and SHOX2 were incubated in 1% FBS in PBST overnight at 4°C. After washing five times for 5 minutes with PBST, appropriate secondary antibodies were added in PBST for 1 h at RT. After washing five times for 5 minutes with PBST, Hoechst 33258 was incubated at the final concentration of 5 μg/ml in DPBS for 15 min at RT. After washing twice with DPBS, cells were mounted with coverslips using fluorescence mounting medium (Dako, S3023) and stored at 4°C until imaging with an inverted confocal microscope (Leica DMi8, Leica Microsystems, Wetzlar, Germany). Primary and secondary antibodies are listed in Supplementary Table S2 .
5
Immunocytochemistry of Ciliated NIH/3T3 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NIH/3T3 cells were seeded on coverslips. Cells were fixed with 4% paraformaldehyde (P6148, Sigma-Aldrich) at room temperature for 15 min. Fixed cells were washed with PBS three times. Then, cells were incubated overnight with primary antibodies in permeabilizing solution (1% bovine serum albumin, and 0.2% triton X-100 in PBS) at 4°C. After washing cells, fluorescence-conjugated secondary antibodies were added and cells were incubated at room temperature for 1 h. Nuclei were stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI, D9542, Sigma-Aldrich). The coverslips were mounted on slide glasses using fluorescent mounting medium (S3023, Dako, CA, USA). Antibodies used for immunocytochemistry are given in Supplementary Table 3 . Images were obtained using a confocal microscope (LSM700, Carl Zeiss, Oberkochen, Germany) equipped with 40x/1.2 (water-immersed) and 63x/1.4 (oil-immersed) objective lenses. Image acquisition was performed using the ZEN software (Carl Zeiss). To calculate the number of ciliated cells, nine non-overlapping fields were selected randomly, and at least 100 cells were counted. Brightness and contrast of images were adjusted using Photoshop CC 2015 (Adobe, CA, USA).
6
Spinal Cord Cryosectioning and Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The spinal cord segments from C3 to T3 were dissected, cryoprotected in 30% sucrose in 0.1 M PBS pH 7.4 for 48 h and embedded in OCT compound (ProSciTech Pty Ltd, QLD, Australia). Spinal cord sections were cut transversely at 10 µm on a cryostat (Leica CM 1950 Cryostat, Amtzell, Germany). Sections were thawed in a 37 °C oven for 10 min, then washed twice for 10 min in Tris phosphate buffered saline (TPBS = 0.05 M PBS + 0.01 M TRIS). The sections were then treated with 50% ethanol/TPBS for 20 min, followed by three 10 min washes in TPBS. DAPI (1 µg/mL) was applied to each slide, incubated for 1 min to visualize the cell nuclei and then washed twice for 10 min. The sections were then coverslipped with fluorescence mounting medium (DAKO, S3023, Carpinteria, CA, US). The sections were imaged with a Zeiss Axio Imager Z2 microscope (Carl Zeiss Microimaging GmbH, Germany). Images were acquired from C3 to T3 for quantitative image analysis. All images were taken at 20× magnification and exposure times were kept constant.
7
Immunostaining of Kidney Biopsy Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All DKD cases were diagnosed based on kidney biopsies performed at the National Clinical Research Center of Kidney Diseases, Medical School of Nanjing University. Kidney biopsies from 5 DKD patients and 6 MCD patients, were obtained from this center’s kidney biorepository. The frozen sections were prepared by cutting the tissue into 5 μm sections with a cryostat (Leica, CM1950) and kept at –80 °C until use. For staining, the sections were washed 3 times in PBS at room temperature followed by blocking buffer (5% BSA) for 30 min at room temperature. Primary antibodies for FXR (Proteintech, 25,055–1-AP) and CREB5(Bioss, bs-14053R) were diluted in PBS and applied to the section followed by incubation at 4 °C overnight. The next day, the slides were washed 3 times for 10 min with TBST, and then a secondary antibody solution was applied and incubated for 1 h at room temperature, washed 3 times for 10 min with TBST. Then slides were stained with DAPI for 10 min at room temperature and washed 3 times for 10 min with TBST and briefly washed with water before mounting (Dako, S3023). The staining images were taken using identical imaging parameters with a confocal laser microscope (Zeiss). Staining intensities and the distribution patterns were compared under the same conditions.
8
Tau Immunohistochemistry of Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin-fixed brains were rinsed in phosphate-buffered saline (PBS) and cryoprotected in 20% sucrose/PBS overnight. The tissue was frozen in optimal cutting temperature (OCT) compound and cut into 25-μm-thick floating sections on a freezing microtome. The sections were immersed in 1% bovine serum albumin/PBS for 30 min and then incubated in anti-PHF tau antibody (AT8, mouse, monoclonal, 1:700; Thermo Fisher Scientific, Tokyo, Japan) for 24 hours at room temperature (RT). After washing in PBS for 30 min, sections were incubated in an anti-mouse secondary antibody conjugated to Q-dot 655 (QD 655) (goat, 1:100 to 1:800, diluted in PBS; Invitrogen, Carlsbad, CA) for 8 hours at RT. A QD 655 dilution at 1:400 (Additional file 1 : Figure S1) for a CBD pretangle provided appropriate immunoEM labeling on tau-positive filaments, whereas its fluorescent signal was not intense enough to delineate subcellular details under confocal microscopy (Additional file 1 : Figure S1). Therefore, the QD-labeled sections were subsequently incubated in an anti-mouse secondary antibody conjugated to Alexa 488 (goat, 1:200; Molecular Probe) for 3 hours at RT to allow more detailed LM observation. After incubation, sections were rinsed in PBS and then mounted in fluorescence-mounting medium (S3023; Dako, Glostrup, Denmark).
9
Quantification of Colonic Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell apoptosis was detected in colonic tissues with a Click-iT™ Plus TUNEL Assay (#C10617; Thermo Fisher Scientific) using the manufacturer’s protocol. The apoptotic bodies were co-localised with cleaved Casp-3 by applying a sequential protocol as reported earlier [40 (link),41 (link)]. After completing the TUNEL protocol, anti-cleaved Casp-3 rabbit IgG monoclonal antibodies (#9661; Cell Signaling Technology Inc.; Danvers, MA, USA) were added at a 1:400 concentration, and the slides were incubated for 3 h. Donkey anti-rabbit IgG antibodies tagged with Alexa Fluor™ 555 (#A-31572; Thermo Fisher Scientific) were then added for 30 min followed by DAPI counterstaining (#D3571; Thermo Fisher Scientific). The slides were cover-slipped with a permanent fluorescence mounting medium (#S3023; Dako, CA, USA) and observed on a Leica DMi8 microscope at 400× magnification. Images were acquired from 15 non-overlapping fields/section, and the apoptotic cells’ numbers and cleaved Casp-3 staining intensity were measured by ImageJ software as previously reported [39 (link),42 (link)].
10
Immunohistochemical Staining of Microglia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Free-floating coronal sections were washed with TBS, permeabilized with 0.5% Triton in TBS and blocked with 1% bovine serum albumin under shaking condition at room temperature for 2 h. Sections were incubated with primary antibodies anti-Iba1 (rabbit polyclonal; C-terminus Iba1; 1 : 1000; Wako 019–19741), anti-CD68 (rat monoclonal, macrosialin protein; 1 : 500, Bio-Rad MCA1957) or antimouse IgG Dy light 405 (donkey polyclonal, IgG (H+L); 1 : 200, Jackson ImmunoResearch N715–475–150) overnight at 4°C. After washing, the sections were incubated with secondary antibodies including donkey antirabbit Alexa Fluor 488 [donkey polyclonal, IgG (H+L); 1 : 500, Thermo Fisher A21206, 2 h at RT] or donkey antirat biotin (Biotin-SP AffiniPure, donkey polyclonal, IgG (H+L); 1 : 400, Jackson Immunoresearch 712–065–150, 2 h at RT), followed by conjugation with streptavidin Alexa Fluor 647 (1 : 500, Thermo Fisher S32357, 1.5 h at RT). Brain sections were mounted on gelatin-coated microscopic slides using fluorescence mounting medium (Dako S3023).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!