Brain slices (PVN and RVLM) were fixed in 4% PB PFA for 24 hours following which the tissue was moved to 4% PB PFA containing 30% Sucrose (cryopreservation). Subsequently, free floating cryostat sections (40 µm thick) were obtained in 10 mM PBS (pH 7.4) and washed (3 times) in PBS. In order to block non-specific binding, sections were incubated in normal goat serum (Vector Labs) prepared in 10mM PBS (with 0.03% Triton-X 100) at room temperature for 1h. Sections were washed three times in 10mM PBS following which they were incubated in primary antibody solution [Rabbit Anti-Tyrosine Hydroxylase (1:500; Abcam-ab6211), normal goat serum, 0.03% Triton-X 100, 10mM PBS-pH 7.4] overnight at 4°C. The sections were washed three times in 10mM PBS and incubated in secondary antibody (Goat anti Rabbit Alexa-568) solution at room temperature for 1h. Subsequently, the sections were washed three times and incubated in the conjugated second primary antibody (Anti-NeuN Alexa-488 conjugate; Abcam-ab190195) at room temperature for 2 hours at a dilution of 1:300. Labeled sections were washed and mounted on gelatin coated slides with plain anti-fade mounting medium (Vector Labs).
Ab190195
Manufactured by Abcam
Ab190195 is a rabbit polyclonal antibody that recognizes human PD1. It is suitable for use in multiple applications including Western blot, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Ab6211, by Abcam (1 mentions)
Rnasein, by Thermo Fisher Scientific (1 mentions)
Superasin, by Thermo Fisher Scientific (1 mentions)
Iodixanol gradient, by Merck Group (1 mentions)
Iscdd, by Roche (1 mentions)
Ab49874, by Abcam (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Reddot, by Biotium (1 mentions)
Ab23703, by Abcam (1 mentions)
6 protocols using ab190195
1
Dual Immunohistochemistry of PVN and RVLM
2
Isolation of Neuronal Nuclei from Human Prefrontal Cortex
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Anonymized post-mortem human tissue was obtained from approved tissue banks and used in accordance with the Human Tissue Act (UK) and with approval by the Imperial College London Research Ethics Committee. Isolation of nuclei was performed as previously described62 (link) with minor modifications. Briefly, 50–250 mg of pre-frontal cortical grey matter was homogenized in 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris pH 8.0, 1 μM DTT, 1X Proteinase Inhibitor w/o EDTA (Roche), 0.4 U μl-1 RNaseIn (ThermoFisher) 0.2 U μl-1 Superasin (ThermoFisher), 1 μM (DAPI) and centrifuged through an iodixanol gradient (Sigma). Pelleted nuclei were washed and then stained with NeuN antibody (Abcam, ab190195) in staining buffer (PBS, 1%BSA, 0.2 U/μl RNaseIn (Thermofisher)), NeuN antibody (1:200) for 1 h at 4 oC. For negative controls antibody was excluded. Nuclei were then sorted on BD Fusion, and collected in wash buffer (PBS, 1%BSA, 0.2 U/μl RNaseIn (Thermofisher)) before RNA extraction.
3
Neuronal Nuclei Isolation and FACS Sorting
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Nuclei were isolated from frozen tissue as described above. Before FACSing, nuclei were incubated with recombinant Alexa Fluor 488 anti-NeuN antibody [EPR12763]–neuronal marker (catalog no. ab190195, Abcam, RRID:AB_2716282) at a concentration of 1:500 for 30 min on ice as previously described (79 (link)). The nuclei were run through the FACS at 4°C with a low flow rate using a 100-mm nozzle, and 300,000 Alexa Fluor 488–positive nuclei were sorted. The sorted nuclei were pelleted at 1300g for 15 min and resuspended in 1 ml of ice-cold nuclear wash buffer (20 mM Hepes, 150 mM NaCl, 0.5 mM spermidine, 1× cOmplete protease inhibitors, 0.1% bovine serum albumin) and 10 μl per antibody treatment of ConA-coated magnetic beads (Epicypher) added with gentle vortexing (pipette tips for transferring nuclei were precoated with 1% bovine serum albumin). Protocol can be found at DOI: dx.doi.org/10.17504/protocols.io.4r3l27pejg1y/v1 .
4
Quantification of Apoptosis in Traumatic Brain Injury
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Apoptosis in 8‐μm frozen brain sections was examined by TUNEL staining according to the manufacturer's directions (ISCDD, Boehringer Mannheim). Brain sections were incubated with 50 μL TUNEL reaction mixture for 1 hour at ambient temperature in a room without light. After the incubating process, the slides were rinsed three times with PBS (pH 7.4) and stained with DAPI to detect nuclei with blue fluorescence. Similarly, NeuN was stained with Anti‐NeuN antibody (1:200, ab190195; Abcam) and ascertained by green fluorescence. By TUNEL assay, apoptotic cells displayed red fluorescence. Separated TUNEL‐positive cells underwent counting process in 1 mm2 field in the trauma area of the five mice sampled randomly from each group. An observer blinded to the research designing process counted TUNEL‐positive cells’ number in each of the 20 consecutive fields of view in five sections of each mouse, and the average number of TUNEL‐positive cells/NeuN in the field of view was calculated for each mouse.
5
Neuronal Nuclei Isolation and FACS Sorting
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Nuclei were isolated from frozen tissue as described above. Before FACSing, nuclei were incubated with recombinant Alexa Fluor 488 anti-NeuN antibody [EPR12763]–neuronal marker (catalog no. ab190195, Abcam, RRID:AB_2716282) at a concentration of 1:500 for 30 min on ice as previously described (79 (link)). The nuclei were run through the FACS at 4°C with a low flow rate using a 100-mm nozzle, and 300,000 Alexa Fluor 488–positive nuclei were sorted. The sorted nuclei were pelleted at 1300g for 15 min and resuspended in 1 ml of ice-cold nuclear wash buffer (20 mM Hepes, 150 mM NaCl, 0.5 mM spermidine, 1× cOmplete protease inhibitors, 0.1% bovine serum albumin) and 10 μl per antibody treatment of ConA-coated magnetic beads (Epicypher) added with gentle vortexing (pipette tips for transferring nuclei were precoated with 1% bovine serum albumin). Protocol can be found at DOI: dx.doi.org/10.17504/protocols.io.4r3l27pejg1y/v1 .
6
Multimodal Protein Imaging in Brain Tissue
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Briefly, 15-μm-thick sections were incubated with a blocking buffer (1× PBS/5% normal goat serum/0.3% Triton X-100) for 1 h. The sections were incubated with the following primary antibodies overnight in PBS at 1:500 dilution factors at 4°C: CB1 (ab23703, abcam) were conjugated with CF™405S Dye (Biotium, Shanghai), glial fibrillary acidic protein (GFAP, ab49874, abcam, conjugated with Cy3), neuronal nuclei (NeuN, ab190195, abcam, conjugated with Alexa Fluor 488) and BDNF (bs-4989R-A647, Bioss, conjugated with Alexa Fluor 647). After washing with PBS, RedDot (Biotium, Shanghai) and DAPI were used for nuclei staining. Later, the sections were observed under a Leica (Wetzlar, Germany) TCS SP5 confocal microscope.
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