Dissected hippocampal tissues were homogenized in RIPA buffer (1% NP‐40, 0.1% SDS, 0.5% PMSF, and 4% Roche protease inhibitor) by sonication. After centrifugating at 12,000g for 20 min at 4°C, the supernatant was collected and protein concentration was measured. A total cell extract containing ~30 μg of protein was subjected to SDS‐PAGE and then transferred onto NC membrane. The membrane was blocked by incubation with 5% skim milk for 90 min and then hybridized overnight with primary antibodies at 4°C. The primary antibodies used include anti‐ferritin light chain (ab109373, Abcam), anti‐ferritin heavy chain (ab183781, Abcam), anti‐TfR1 (13–6800, Invitrogen), anti‐Bcl2 (12789‐1‐AP, Proteintech), anti‐Bax (50599‐2‐Ig, Proteintech), anti‐brain‐derived neurotrophic factor (BDNF) (ab108319, Abcam), anti‐furin (ab183495, Abcam), and anti‐β‐actin (CW0096, CWBIO). After washing with tris‐buffer saline with 0.05% Tween‐20, the membrane was incubated at room temperature for 90 min with anti‐rabbit or anti‐mouse secondary antibody conjugated with horseradish peroxide (Zhongshan Biotechnology). Immunoreactive bands were detected by the enhanced chemiluminescence detection kit (Amersham, UK) and visualized by a Bio‐Rad chemiluminescence imager.
Ab183495
Manufactured by Abcam
Sourced in United Kingdom
Ab183495 is a primary antibody raised against a specific target. It is designed for use in research applications, but its detailed function and intended use are not provided in this factual and unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
Ab92323, by Abcam (2 mentions)
Pipa529118, by Thermo Fisher Scientific (2 mentions)
Ls c150813 1, by LSBio (2 mentions)
Ab200997, by Abcam (2 mentions)
Nb100 56576, by Novus Biologicals (2 mentions)
Chemiluminescence imager, by Bio-Rad (1 mentions)
Anti bcl2 12789 1 ap, by Proteintech (1 mentions)
Enhanced chemiluminescence detection kit, by Cytiva (1 mentions)
Cw0096, by CWBIO (1 mentions)
Ab109373, by Abcam (1 mentions)
Ab183781, by Abcam (1 mentions)
Ab108319, by Abcam (1 mentions)
Z0334, by Agilent Technologies (1 mentions)
Alexa fluor 488 or 594 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Af 556 na, by R&D Systems (1 mentions)
Mab360, by Merck Group (1 mentions)
Ma1 045, by Thermo Fisher Scientific (1 mentions)
Bz x700, by Keyence (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Ab108252, by Abcam (1 mentions)
8 protocols using ab183495
1
Hippocampal Protein Expression Analysis
2
Immunofluorescence Analysis of Brain Proteins
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In brief, brain sections were fixed (ice cold 4% paraformaldehyde), washed, permeabilized (0.3% Triton X‐100 in PBS) and blocked with appropriate non‐immune sera. After that, specimens were incubated (overnight, 4◦C) with primary antibodies against mouse anti‐PSD95 (1:500, mouse, MA1‐045, Thermo Fisher), anti‐GFAP (1:500, MAB360; Millipore), rabbit anti‐GFAP (1:5000, Z0334; Dako), anti‐GluN2A (1:500, NBP2‐19551, Novus Biologicals), anti‐furin (1:500, ab183495; abcam), anti‐SNAP23 (1:500, 10825‐1‐AP; proteintech), anti‐VAMP3 (1:500, 10702‐1‐AP; proteintech), anti‐pNF‐κB (1:500, 3033; CST), anti‐synaptophysin (1:500, 36406; CST), the goat anti‐β‐NGF (1:200, AF‐556‐NA; R&D Systems). The secondary antibodies used in the present experiments were Alexa Fluor®‐488 or −594 conjugated goat anti‐mouse IgG, and Alexa Fluor®‐594 or‐488 conjugated goat anti‐rabbit IgG or donkey anti‐goat Alexa Fluor®‐594 conjugated donkey anti‐goat IgG (1:500, Invitrogen). Cell nuclei were visualized with Hoechst 33342 (1:1000) or DAPI. Fluorescence‐labeled optical sections were captured with a Leica confocal laser scanning microscope (TCS SP5). Image J software (W.S. Rasband, U. S. National Institutes of Health, Bethesda, Maryland, USA) was used for immunofluorescent intensity quantitative analysis of brain sections.
3
Immunohistochemical Profiling of SARS-CoV-2 Entry Factors
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Immunohistochemical staining was performed for Ace2 and Tmprss2.
Four-micrometer-thick serial paraffin-embedded sections were deparaffinized in xylene and dehydrated in ethanol and then treated with 3% H2O2 to block endogenous peroxidase activity and incubated with Blocking One (Nacalai Tesque, 34 Kyoto, Japan) to block non-specific immunoglobulin binding prior to immunostaining. After antigen activation, the tissue sections were probed with primary anti-Ace2 (1:300 dilution; rabbit monoclonal, Abcam, ab108252; Cambridge, UK), anti-Tmprss2 (1:1000 dilution; rabbit monoclonal, Abcam, ab92323; Cambridge, UK), and anti-Furin (1:100 dilution; rabbit monoclonal, 1 Abcam, ab183495; Cambridge, UK) antibodies, followed by probing with appropriate peroxidase-conjugated secondary antibodies and a (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted June 23, 2020. ; https://doi.org/10.1101/2020.06.23.164335 doi: bioRxiv preprint 7 diaminobenzidine substrate. Images of all sections were captured using a digital microscope camera (Keyence BZ-X700) with 4×, 10×, and 40× objective lenses.
Four-micrometer-thick serial paraffin-embedded sections were deparaffinized in xylene and dehydrated in ethanol and then treated with 3% H2O2 to block endogenous peroxidase activity and incubated with Blocking One (Nacalai Tesque, 34 Kyoto, Japan) to block non-specific immunoglobulin binding prior to immunostaining. After antigen activation, the tissue sections were probed with primary anti-Ace2 (1:300 dilution; rabbit monoclonal, Abcam, ab108252; Cambridge, UK), anti-Tmprss2 (1:1000 dilution; rabbit monoclonal, Abcam, ab92323; Cambridge, UK), and anti-Furin (1:100 dilution; rabbit monoclonal, 1 Abcam, ab183495; Cambridge, UK) antibodies, followed by probing with appropriate peroxidase-conjugated secondary antibodies and a (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted June 23, 2020. ; https://doi.org/10.1101/2020.06.23.164335 doi: bioRxiv preprint 7 diaminobenzidine substrate. Images of all sections were captured using a digital microscope camera (Keyence BZ-X700) with 4×, 10×, and 40× objective lenses.
4
Protein Extraction and Western Blotting Analysis
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A RIPA protein extraction kit (Beyotime Biotechnology China) containing phenylmethylsulfonyl fluoride (PMSF; Beyotime Biotechnology China) was used to extract total protein. An enhanced bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology) was used to determine protein concentrations. Western blotting analysis was performed according to published protocols.19 (link) The following antibodies were used: furin (1:1000, Abcam, ab183495), S100 calcium-binding protein B (S100β) (1:1000, Abcam, ab52642), SRY-Box transcription factor 9 (SOX9) (1:5000, Abcam, ab185966), polypyrimidine tract binding protein 1 (PTBP1) (1:2000, Proteintech, 12582-1-AP), glial fibrillary acidic protein (GFAP) (1:2000, Proteintech, 16825-1-AP), adenylate cyclase 3 (ADCY3) (1:1000, Proteintech, 19492-1-AP), Rab28 (1:1000, Thermo, PA5-68303), NeuN (1:1500, Abcam, ab177487), GAPDH (1:5000, Proteintech, 10494-1-AP), and horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (1:3000, Proteintech). The bands were visualized using Western Bright ECL (Advansta, US) and a Fusion FX5 image analysis system (Vilber Lourmat, France).
5
Immunohistochemical Analysis of COVID-19 Tissues
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4-μm-thick sections were cut from paraffin blocks of lung and liver tissues from COVID-19 patients and non-COVID-19 donors. IHC staining with an anti-FXa protein antibody (PIPA529118, Invitrogen), an anti-furin antibody (ab183495, Abcam), an anti-trypsin antibody (ab200997, Abcam), or an anti-plasmin antibody (LS-C150813–1, LSBio) as a primary antibody was performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute. Stained slides were mounted and scanned for observation.
Mouse tissues isolated from experimental mice were placed in 10% neutral buffered formalin for a minimum of 72 hours. After paraffin embedding, 4-μm-thick sections were cut from the blocks. H&E staining and IHC with anti-NP protein antibody (NB100–56576, Novus) as the primary antibody were performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute. Stained slides were mounted and scanned for observation.
Mouse tissues isolated from experimental mice were placed in 10% neutral buffered formalin for a minimum of 72 hours. After paraffin embedding, 4-μm-thick sections were cut from the blocks. H&E staining and IHC with anti-NP protein antibody (NB100–56576, Novus) as the primary antibody were performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute. Stained slides were mounted and scanned for observation.
6
Quantifying SARS-CoV-2 Entry Factors
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Lungs, heart chambers and kidneys were homogenized on ice and centrifuged at 4°C for 5 minutes at 1000 g. The homogenized tissue was then lysed in RIPA buffer (150 mmol/L NaCl, 1% NP40, 50 mmol/L Tris pH 8.0, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with a cocktail of protease inhibitors (Roche) in rotation at 4°C for 20 minutes, and then centrifuged at 4°C for 10 minutes at 13300 g. The cleared supernatant was collected, and protein concentration was determined (Bradford reagent, Sigma). Equal amounts of extracted proteins (40‐60 μg) were loaded and run on a 9% SDS‐polyacrylamide gel and were transferred to nitrocellulose membrane. Membranes were incubated in blocking buffer, TBS‐T (Tris‐buffered saline, 0.1% Tween 20) containing 5% (w/v) BSA, and probed with the appropriate primary antibodies: anti‐ACE2 (1:1000, goat, AF933, R&D Systems), anti‐Furin (1:1000, rabbit, ab183495, Abcam), anti‐TMPRSS2 (1:500, rabbit, ab92323, Abcam), anti‐ADAM17 (1:1000, rabbit, GTX101358, GeneTex) and anti‐GAPDH (1:500, mouse, sc‐32233, Santa Cruz). After washing with TBS‐T, the immunoreactive proteins were visualized with horseradish‐conjugated goat anti‐rabbit (1:25 000, 111‐035‐144, Jackson), donkey anti‐mouse (1:10 000, 715‐035‐151, Jackson) and donkey anti‐goat (1:10 000, 705‐035‐003, Jackson) IgG secondary antibodies and chemiluminescent substrate.
7
Histological Analysis of COVID-19 Tissues
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4-μm-thick sections were cut from paraffin blocks of lung and liver tissues from COVID-19 patients and non-COVID-19 donors. The following primary antibodies were used for IHC staining: an anti-FXa protein antibody (PIPA529118, Invitrogen, at a 1:500 dilution), an anti-furin antibody (ab183495, Abcam, at a 1:500 dilution), an anti-trypsin antibody (ab200997, Abcam, at a 1:500 dilution), or an anti-plasmin antibody (LS-C150813-1, LSBio, at a 1:500 dilution). IHC was performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute. Stained slides were mounted and scanned for observation.
Tissues isolated from the experimental mice were placed in 10% neutral buffered formalin for a minimum of 72 hours. After paraffin embedding, 4-μm-thick sections were cut from the blocks. H&E staining and IHC with an anti-NP protein antibody (NB100-56576, Novus, at a 1:500 dilution) as the primary antibody were performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute. Stained slides were mounted and scanned for observation.
Tissues isolated from the experimental mice were placed in 10% neutral buffered formalin for a minimum of 72 hours. After paraffin embedding, 4-μm-thick sections were cut from the blocks. H&E staining and IHC with an anti-NP protein antibody (NB100-56576, Novus, at a 1:500 dilution) as the primary antibody were performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute. Stained slides were mounted and scanned for observation.
8
Protein Expression Analysis of HUVECs
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Protein lysates of HUVECs were prepared using Radio Immuno Precipitation Assay (RIPA) Lysis Buffer. Western blot analysis was performed using antibodies against FURIN (Abcam; ab183495), endothelin‐1 (Abcam; ab117757), vascular cell adhesion molecule‐1 (VCAM1) (Abcam; ab174279), MCP1 (monocyte chemotactic protein‐1) (Abcam; ab151538), and beta‐Actin (Sangon Biotech; D110001).
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