Hippocampal slices were washed 3 times with 0.01 M phosphate buffer saline (PBS) and fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature (RT). At the end of fixation, the slices were washed 3 times with PBS and then stored at 4 °C in PBS containing 0.05% sodium azide until use. Prior to immunolabelling, slices were washed 3 times with PBS for 10 min, then incubated overnight with 1% Triton X-100 in PBS at 4 °C, followed by a blocking stage using 20% bovine serum albumin (BSA) diluted in PBS (0.01 M) and containing 0.1% Triton (PBS-T) for 3 h at RT. Slices were then incubated overnight with primary antibody diluted in PBS-T and 1% normal goat serum (NGS): rabbit anti-NeuN antibody (1: 200, Cell Signalling Technology, D4G4O # 24,307), guinea pig anti-GLT1 antibody (1:5000, Merck Millipore Chemicon International, ab1783), rabbit anti-GLAST antibody (1: 150, Abcam, ab416), or rabbit anti-GS (1:5000, Abcam, ab49873). Following washes in PBS-T, slices were incubated in appropriate secondary antibodies for 3 h at RT, namely goat anti-guinea pig Alexa-fluor 568 or goat anti-rabbit Alexa-Fluor 647 (both at 1:500, Invitrogen), in addition to the nuclear chromatin dye Hoechst 33,342 (1:500, Fisher, 11,544,876). Lastly, slices were washed in PBS-T (0.1%) and mounted in Fluoromount G (Invitrogen—REF 00–4958-02).
Ab49873
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab49873 is a laboratory equipment product offered by Abcam. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Tissue tek, by Sakura Finetek (3 mentions)
Ab416, by Abcam (2 mentions)
Dm5500 widefield microscope, by Leica (2 mentions)
Rnascope multiplex fluorescent v1 assay, by Advanced Cell Diagnostics (2 mentions)
Mm per1, by Advanced Cell Diagnostics (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti guinea pig, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti neun antibody, by Cell Signaling Technology (1 mentions)
Ab1783, by Abcam (1 mentions)
Envision system hrp dab, by Agilent Technologies (1 mentions)
Laser confocal microscope, by Leica (1 mentions)
A28180, by Thermo Fisher Scientific (1 mentions)
Ab104139, by Abcam (1 mentions)
Tissue tek, by Ted Pella (1 mentions)
Ab66155, by Abcam (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Ab5535, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
11 protocols using ab49873
1
Immunolabeling of Hippocampal Slices
2
Liver Tumor Histopathological Analysis
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Mice were sacrificed shortly after the last MRI acquisition. All livers were macroscopically analyzed for focal hepatic nodules. Before liver sampling, the orientation of all liver lobes, relative to the longitudinal, transversal and sagittal axis of the animal, was marked, and the size and location of the macroscopic tumor nodes were measured with a caliper and correlated with the MRI findings. Part of the respective liver was then embedded in paraffin and used for diagnostic procedures. The rest of the liver was frozen for further analysis.
All tissue samples were fixed in 10% buffered formalin for 24 h and paraffin embedded. For histological analysis, 4-μm-thick serial sections were stained with Hematoxylin and Eosin. Reticulin was visualized by Gomori staining. Immunostaining was performed with anti-GS (Zulehner et al., 2010 (link)) (1:500; ab49873, Abcam, Cambridge, UK) and anti-SAA (1:100; PAB795Mu01, Cloud-Clone, Houston, TX, USA) antibodies. Reactions were developed using an EnVision+ System-HRP (DAB) (DAKO, Carpinteria, CA, USA). After immunostaining, slides were counterstained with Hematoxylin. Positive and negative controls were included for each run.
All tissue samples were fixed in 10% buffered formalin for 24 h and paraffin embedded. For histological analysis, 4-μm-thick serial sections were stained with Hematoxylin and Eosin. Reticulin was visualized by Gomori staining. Immunostaining was performed with anti-GS (Zulehner et al., 2010 (link)) (1:500; ab49873, Abcam, Cambridge, UK) and anti-SAA (1:100; PAB795Mu01, Cloud-Clone, Houston, TX, USA) antibodies. Reactions were developed using an EnVision+ System-HRP (DAB) (DAKO, Carpinteria, CA, USA). After immunostaining, slides were counterstained with Hematoxylin. Positive and negative controls were included for each run.
3
Zonal Expression of Clock Genes in Liver
4
Immunohistochemical Localization of SHP-1, pJNK, and GS
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The eyecups were fixed in 4% paraformaldehyde for 1 hour and dehydrated sequentially in 20% and 30% sucrose solutions at 4°C for 1 hour. After the anterior sections had been removed and the cups filled with optimal cutting temperature compound (Tissue‐Tek; Ted Pella), they were then frozen at −80°C, and cut in the sagittal direction (8‐μm‐ thick sections). After being blocked with 5% goat serum and permeated with 0.15% Triton X‐100 in PBS for 45 minutes, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti‐SHP‐1(sc‐7289, diluted 1:50; Santa Cruz Biotechnology); mouse anti‐pJNK (9255S, diluted 1:100; Cell Signaling Technology) and rabbit anti‐ glutamate synthase (GS) (ab49873, diluted 1:5000; Abcam). The sections were rinsed three times with PBS and incubated with a secondary antibody (A28180; Invitrogen; or 4414, Cell Signaling Technology) for 1 hour at room temperature. After the sections were rinsed three times, they were counterstained with Fluoroshield with DAPI mounting medium (ab104139; Abcam). The sections were then observed with a laser confocal microscope (Leica Microsystems).The immunofluorescence intensity in the images was measured with ImageJ software (National Institutes of Health).
5
Quantitative Liver Histopathology
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Liver samples were formalin‐fixed, paraffin‐embedded, sectioned at 4 µm, and processed routinely for H&E staining. Immunohistochemical staining of glutamine synthetase (ab49873; 1:10 000; Abcam) and Ki67 (ab66155; 1:1000; Abcam) was performed on formalin‐fixed, paraffin‐embedded liver sections with the Histar Detection Kit Star3000a according to the manufacturer's instructions (Bio‐Rad Laboratories, Inc). Morphometry was used to quantify the stained tissue area using ImageJ software (National Institute of Health). Slides were counterstained with hematoxylin.
6
Mouse Eye Tissue Preparation and Immunostaining
7
Zonal Expression of Clock Genes in Liver
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smFISH of R genes were done on fresh-frozen liver cryosections (8μm) embedded in O.C.T Compound (Tissue-Tek; Sakura-Finetek USA), sampled every three hours (ZT0 to ZT21). RNAscope® probes for Bma1l mRNA (Mm-Arntl, catalog #: 438748-C3) and Per1 mRNA (Mm-Per1, catalog #: 438751) were used, according to the manufacturer’s instructions for the RNAscope Fluorescent Multiplex V1 Assay (Advanced Cell Diagnostics). To detect the central vein, an immunofluorescence of Glutamine Synthetase (ab49873, Abcam, diluted 1:2000 in PBS/BSA 0.5%/Triton-X0.01%) was done together with smFISH. Nuclei were counterstained with DAPI and sections were mounted with ProLong™ Gold Antifade Mountant. Liver sections were imaged with a Leica DM5500 widefield microscope and an oil-immersion x63 objective. Z-stacks were acquired (0.2μm between each Z position) and mRNA transcripts were quantified using ImageJ, as described previously in ref.50 (link). Pericentral (PC) and Periportal (PP) veins were manually detected based on Glutamine Synthetase IF or on bile ducts (DAPI staining). The Euclidean distance between two veins and the distance from the vein of each mRNA transcript were calculated. mRNA transcripts were assigned to a PP or PC zone if the distance from the corresponding vein was smaller than one-third of the distance between the PP and PC veins (ranging from 50 to 130μm).
8
Immunohistochemical Analysis of Liver Vasculature
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Mouse liver tissues were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin, as we previously reported.[22 , 23 ] Sections of 3.5‐µm thickness were incubated in citrate buffer (pH 6.0, BOSTER AR0024) for 15 min at 120ºC, and the endogenous peroxidase was blocked by 3% H2O2 for 10 min. The slides were incubated with 10% bovine serum albumin in PBS for 30 min at 37ºC to block the nonspecific binding sites, followed by incubation with appropriate primary antibodies for overnight at 4ºC, and then with horseradish peroxidase anti‐rabbit IgG or anti‐mouse IgG antibodies for 30 min. Color was then developed by incubation with DAB Substrate kit (ORIGENE ZLI‐9019). After washing in PBS, tissue sections were counterstained with hematoxylin and viewed under a microscope. Major primary antibodies used in the study include anti‐CD31 antibody (1:100; 77699s, CST), anti‐GS antibody (1:2000; ab49873, Abcam),[24 , 25 , 26 , 27 ] anti–lymphatic endothelial receptor‐1 (LYVE1) antibody (1:4000; ab281587, Abcam), and anti–α‐smooth muscle actin (α‐SMA) antibody (1:800; A2547, Sigma).
CD31+ portal vessels (excluding diameters ≤30 µm) and CD31+ central veins were manually counted based on staining of CD31 and GS. Six to eight randomly chosen fields from each section were analyzed.
CD31+ portal vessels (excluding diameters ≤30 µm) and CD31+ central veins were manually counted based on staining of CD31 and GS. Six to eight randomly chosen fields from each section were analyzed.
9
Trigeminal Ganglion Immunostaining Protocol
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Six to 8 microns-thick frozen sections of trigeminal ganglion were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton and subsequently blocked with Blocking One Histo (Nacalai Tesque, Kyoto, Japan). Sections were incubated with primary antibodies, rabbit anti-glutamine synthetase (1:1000; ab49873, Abcam, Cambridge, UK) and goat anti-GFAP (1:500; ab53554, Abcam), overnight at 4 °C. Sections were incubated with secondary antibodies for 2 h at room temperature using donkey anti-goat IgG Alexa Fluor 488 (1:200; ab150129, Abcam) and donkey anti-rabbit Alexa Fluor 555 (1:200; ab 150074, Abcam). For nuclear staining, 4’, 6-Diamidino-2-phenylindole dihydrochloride (DAPI, 1:100, Nacalai Tesque, Inc., Kyoto, Japan) was used for 15 min at room temperature, followed by mounting with Aqua-Poly/Mount (Polysciences, Inc., Warrington, PA, USA). Isotypes (goat IgG bs-0294P, and rabbit IgG bs-0295P, Bioss Antibodies, Boston, MA, USA) and only secondary antibodies were used as positive and negative control. Images were observed and acquired by confocal laser-scanning microscope (LSM 700, Carl Zeiss, Oberkochen, Germany).
10
Müller Cell Response to LPS and LCN2
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Müller cells were seeded on coverslips in 24-well plates, and then simultaneously stimulated with 1 μg/ml LPS and/or 1 μg/ml LCN2 for 1 h. After stimulation, the cells were washed with PBS and then fixed with 4% paraformaldehyde for 15 min. After the cell membranes were permeabilized with 0.3% Triton X-100, they were blocked with 5% goat serum for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-GS antibody (ab49873, diluted 1:100; Abcam), rabbit anti-NF-κB p65 antibody (4764S, diluted 1:100; Cell Signaling Technology, Beverly, MA, USA), or rabbit anti-NF-κB phospho-p65 antibody (3033S, diluted 1:100; Cell Signaling Technology). The coverslips were then washed three times with PBS and incubated with the corresponding secondary antibodies (A-11008, A-21428, diluted 1:1000, Invitrogen) for 1 hour at room temperature. After three washes with PBS, the cells were counterstained with DAPI (Sigma-Aldrich) for 5 min. The coverslips were visualized and photographed with fluorescence microscopy (Leica Microsystems).
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