Immunohistochemistry was performed to observe the expression of HTT. The anti-HTT antibody (ab109115, Abcam) was incubated overnight at 4°C, followed by incubation with biotinylated anti-rabbit secondary antibodies for one hour and counterstained with hematoxylin to detect HTT expression on human OSCC tissue sections. The number of HTT-positive cells and nucleated cells in five random fields of view on each human OSCC tissue and NCMT tissue section were counted Using ImageJ software. Their ratio (HTT-positive cells/nucleated cells) was used for statistical analysis. Table 1
Ab109115
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab109115 is a primary antibody product. It is designed for use in immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Mab2166, by Merck Group (2 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Affinipure, by Jackson ImmunoResearch (1 mentions)
Sulfo tag, by Mesoscale (1 mentions)
Mab1574, by Merck Group (1 mentions)
Calreticulin, by Abcam (1 mentions)
Anti ctip2, by Abcam (1 mentions)
α tubulin, by Abcam (1 mentions)
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexafluor 680 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Anti darpp32, by Santa Cruz Biotechnology (1 mentions)
A2066, by Merck Group (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ab8245, by Abcam (1 mentions)
Phosstop, by Roche (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
A1978, by Merck Group (1 mentions)
10 protocols using ab109115
1
Immunohistochemical Analysis of HTT Expression in OSCC
2
Antibody Panel for Huntington's Disease
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Primary antibodies employed in this study were: mouse anti-EEA1 (BD Biosciences #610456, IF 1:200), mouse anti-SNX1 (BD Biosciences #611482, IF 1:200), rabbit anti-VPS35 (Abcam #ab97545, IF 1:400), sheep anti-TGN38/46 (Genetex #GTX74290, IF 1:400), mouse anti-CD63 (Santa Cruz #MEM259, IF 1:250), mouse anti-LAMP1 (Hybridoma Bank #H4A3, IF 1:400), rabbit anti-Huntingtin (Abcam #ab109115, IF 1:400 and WB 1:1000), mouse anti-Huntingtin (Thermo Fisher 3-19, IF 1:200) rabbit anti-Septin 2 (Abcam #ab179436, WB 1:1000), rabbit anti-Septin 7 (Proteintech #13818, IF 1:200 and WB 1:1000), rabbit anti-Septin 9 (Proteintech # 10769, IF 1:200 and WB 1:1000), mouse anti-GFP (Roche #11 814 460 001, WB 1:2000), rabbit anti-SNX21 (Proteintech #22193-1-AP, WB 1:1000), mouse anti-rabbit conformation specific (Cell Signaling #3678, WB 1:1000) and rabbit IgG (EPR25A) isotype control (Abcam #172730). Jackson ImmunoResearch Alexa-Fluor680/800-conjugated secondary antibodies were used for quantitative detection of proteins by western blot at 1:18,000 and Invitrogen Alexa-Fluor secondary antibodies for detection of primary antibodies in immunofluorescence experiments at 1:400.
3
Immunohistochemical detection of ANT2
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Human tissue sections were incubated overnight at 4°C with an anti-ANT2 antibody (ab109115; Abcam, Cambridge, MA, United States) and then incubated for 1 h with a biotin-labeled anti-rabbit secondary antibody. The samples were counterstained with hematoxylin. Analysis was performed using a non-biotin polymer HRP detection system (BioGenex Laboratories, San Ramon, CA, United States).
4
Huntingtin Protein Antibody Characterization
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All antibodies used in this study are commercially available: EPR5526, rabbit monoclonal anti-HTT (Abcam Cat# ab109115, RRID:AB_10863082); MW1, mouse monoclonal anti-polyQ (DSHB Cat# mw1, RRID:AB_528290); 1C2, mouse monoclonal anti-polyQ (Millipore Cat# MAB1574, RRID:AB_94263); 3B5H10, mouse monoclonal anti-polyQ (Sigma-Aldrich Cat# P1874, RRID:AB_532270); MAB5492, mouse monoclonal anti-HTT (Millipore Cat# MAB5492, RRID:AB_347723); D7F7, rabbit monoclonal anti-HTT (Cell Signaling Technology Cat# 5656, RRID:AB_10827977); MAB2166, mouse monoclonal anti-HTT (Millipore Cat# MAB2166, RRID:AB_ 2123255); peroxidase-conjugated AffiniPure, donkey polyclonal anti-rabbit IgG (Jackson ImmunoResearch Labs Cat# 711–035–152, RRID:AB_10015282); peroxidase-conjugated AffiniPure, donkey polyclonal anti-mouse IgG (Jackson ImmunoResearch Labs Cat# 715–035–150, RRID:AB_2340770) and Sulfo-Tag labeled, goat polyclonal anti-mouse IgG (Meso Scale Discovery, Cat# R32AC, RRID:AB_2783819).
5
Immunofluorescence and Western Blot Protocols
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Ab109115 (Abcam) was used at a dilution of 1:100, and Mab2166 (Merck Millipore) was used at 1:1000 for immunofluorescence. S421 was a kind gift from Dr. Sandrine Humbert, (Institut Curie, Paris, France), and was used at 1:1000 for immunofluorescence. Anti-EGFR (New England Biolabs) was used at 1:50 for immunofluorescence and 1:1000 for western blots. Calreticulin (Abcam) was used at 1:500 for immunofluorescence, and α-tubulin was used at 1:5000 for western blots. Anti-CTIP2 (Abcam) was used at 1:500, and Anti-DARPP32 (Santa Cruz Biotechnology Inc.) was used at 1:200; both for immunofluorescence. Secondary antibodies; Goat-anti-Rabbit AlexaFluor 568 and Donkey-anti-Mouse AlexaFluor 488 (Life Technologies) were both used at 1:100 for immunofluorescence. Goat-anti-Rabbit AlexaFluor 680 (Life Technologies) and Goat-anti-mouse DyLight 800 were used for Western blots at 1:10,000 and 1:20,000, respectively.
6
Western Blotting of Huntingtin Protein
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For western blotting mice were sacrificed and brains were grinded and shaked in magic mix (48% urea, 15 mM TRIS-HCl (pH7.5), 8.7% glycerin, 1% SDS, 1% mercaptoethanol, complete protease inhibitors (Roche), Phosstop (Roche)) at 4°C with add-on homogenization (QIAshredder). Afterwards, samples were boiled at 95°C and 30 µg protein lysate (40 µg for Htt blot) was loaded on a 10% SDS PAGE gel, resolved (overnight at 100 V for separation of mHtt from wtHtt) and blotted onto a PVDF membrane (BioRad) using TransBlot Turbo (BioRad). Membranes were then blocked with 1% BSA (pS6) or 5% milk (Actin, Gapdh, Htt) in PBS-Tween20 (Roth) and incubated with primary antibody (Actin: 1:2000, A2066, Sigma Aldrich; pS6: 1:2000, 2215, cell signaling; Gapdh: 1:2000, ab8245, abcam; Htt: 1:850, ab109115, abcam) in blocking buffer overnight. Membranes were then washed three times with PBS-Tween20 and subsequently incubated with secondary antibody (1:6000, for Htt 1:4000, Donkey anti-rabbit or anti-mouse, Jackson Immuno Research) for 1 hr in blocking buffer. Subsequently, membranes were again washed three times with PBS-Tween20. Chemiluminescent detection was done by using Western Lightning Plus-ECL (PerkinElmer). Visualisation was performed on a ChemiDoc MP Imaging System (Biorad). Quantification of resulting bands was performed using Image Lab (version 5.2.1).
7
Western Blot Analysis of Mutant HTT Protein
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For analysis of mutant HTT protein levels, fresh frozen striatal samples were collected for Western blot. Protein lysates were loaded on a 3–8% gradient gel (Tris‐acetate Precast Gel, Bio‐Rad) before being transferred onto a polyvinylidene fluoride (PVDF) membrane using the Bio‐Rad Trans‐Blot Turbo Transfer System. Membranes were blotted with rabbit monoclonal primary antisera directed against HTT (1:1000, #AB109115, Abcam), rabbit monoclonal primary antisera directed against PDE10A (1:10,000, #AB227829, Abcam), rabbit polyclonal primary antisera directed against DARPP‐32 (1:1000, #TA309102, Origene) or mouse monoclonal primary antisera directed against beta‐actin (1: 10,000, #A1978, Sigma‐Aldrich). Membranes were then probed with their respective secondary antibodies, developed and analysed with densitometry.
8
Huntingtin Aggregates Immunohistochemistry
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Tissue sections were de-paraffinized and boiled in citrate buffer (pH 8) for 20 min for antigen retrieval, then cooled for 20 min at room temperature (RT). For NII staining, sections were incubated with 25 μg/ml proteinase K at 30 min/37 °C to remove cytosolic HTT. Sections were treated with 0.3% H2O2 for 30 min/RT, then permeabilized with blocking buffer (PBS with 2.5% normal horse serum (NHS)) for 1 h/RT, followed by incubation with primary antibody for 30 min/RT. Sections were washed twice in PBS for 5 min and secondary antibody was incubated for 30 min/RT, followed by 2 × 5 min PBS washes. For immunohistochemistry, sections were colorized using NovaRED (SK-4800, Vector Laboratories, Burlingame, CA). Primary antibodies: Huntingtin 1:100 (ab109115, Abcam, Cambridge, UK); GFAP 1:500 (GA524, Dako Omnis); Iba1 1:200 (019-10741, Wako Chemicals). Fluorescent secondary antibodies: Alexa Fluor 488 donkey anti-rabbit 1:250 (Jackson ImmunoReseach, AB_2313584).
In situ hybridization (ISH) was performed according to the RNAScope Fast-RED protocol followed by autofluorescence quenching for 5 min with TrueView (Vector Laboratories). Counterstaining with DAPI (1:20,000 in PBS with 2.5% NHS) was performed to identify Htt aggregates that are NIIs.
In situ hybridization (ISH) was performed according to the RNAScope Fast-RED protocol followed by autofluorescence quenching for 5 min with TrueView (Vector Laboratories). Counterstaining with DAPI (1:20,000 in PBS with 2.5% NHS) was performed to identify Htt aggregates that are NIIs.
9
Huntingtin Protein Detection by WB
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Antibodies used for SDS-PAGE western blot (WB) analysis: α-Htt (ab109115, Abcam); α-Htt D7F7 (#5656 CST); α-Flag M2 (#SLBV9325, Sigma-Aldrich); α-Vinculin (#V9131, Sigma-Aldrich); α-F8A1/Hap40 (#PA5-61382, Invitrogen), α-AHSA1 (#12841, CST). SDS-PAGE WB was performed according to standard procedures, except for Htt (∼350kDa). For Htt a 6%separating tris-glycine SDS-PAGE gel was used and proteins were run out of the gel until ∼80 kDa. Wet blotting was done O/N at 45V at 4°C. Proteins were visualized using LiCOR secondary antibodies and the Odyssey system.
10
Characterizing Huntington's Disease Protein Dynamics
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The fluorescent proteins and human Httex1, Httex1 TC9 , and Httex1 TC1 as fusions to fluorescent proteins were expressed in vectors with cytomegalovirus (CMV) promoters. The gene encoding the fast fluorescent timer variant of mCherry was synthesized from the reported sequence (Subach et al., 2009) . The GFP-TIA1 construct was kindly provided by Myriam Gorospe (NIA-NIH). The lentiviral vectors FUGW, psPAX2, and pCMV-VSV-G were used to generate lentiviral particles for expressing Httex1 variants.
Cell Culture HEK293T, AD293, and HeLa cells (ATCC) were maintained in DMEM and Neuro2a in Optimem (Invitrogen). The medium was supplemented with 10% (v/v) fetal calf serum (FCS), 1 mM glutamine, and penicillin-streptomycin (pen-strep). The HeLa tet repressor line was generated with the T-REx system (Invitrogen). H9 human embryonic stem cells were induced into the neuronal lineage as described previously (Denham et al., 2012) . The day 7 neurospheres were differentiated into cortical neurons using N2B27 medium supplemented with 1 mM N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and 20 ng/mL brain derived neurotrophic factor (BDNF).
Western Blot AD293 cells were transfected with pT-REx-Httex1(97Q) TC9 -Cerulean. After 24 hr, lysates (30 mg of cellular proteins) were subjected to western blot probed with anti-Huntingtin antibody (Abcam, ab109115).
Cell Culture HEK293T, AD293, and HeLa cells (ATCC) were maintained in DMEM and Neuro2a in Optimem (Invitrogen). The medium was supplemented with 10% (v/v) fetal calf serum (FCS), 1 mM glutamine, and penicillin-streptomycin (pen-strep). The HeLa tet repressor line was generated with the T-REx system (Invitrogen). H9 human embryonic stem cells were induced into the neuronal lineage as described previously (Denham et al., 2012) . The day 7 neurospheres were differentiated into cortical neurons using N2B27 medium supplemented with 1 mM N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and 20 ng/mL brain derived neurotrophic factor (BDNF).
Western Blot AD293 cells were transfected with pT-REx-Httex1(97Q) TC9 -Cerulean. After 24 hr, lysates (30 mg of cellular proteins) were subjected to western blot probed with anti-Huntingtin antibody (Abcam, ab109115).
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