重组Anti-VGluT1抗体[EPR22269]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VGluT1 antibody [EPR22269] (AB227805)

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling VGluT1 with ab227805 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Specific cytoplasmic staining on mouse hippocampus, positive staining was also observed on mouse cerebral cortex and thalamus (PMID : 29532891). Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-VGluT1 antibody [EPR22269] (AB227805)

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-VGluT1 antibody [EPR22269] (AB227805)

IHC image of vGluT1 staining in a section of frozen normal human cerebellum performed on a Leica BOND^TM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab227805, 0.1ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VGluT1 antibody [EPR22269] (AB227805)

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling VGluT1 with ab227805 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Negative control : No staining on human liver is observed.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VGluT1 antibody [EPR22269] (AB227805)

Immunohistochemical analysis of paraffin-embedded human cerebral cortex tissue labeling VGluT1 with ab227805 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on human cerebral cortex (PMID : 29532891). Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-VGluT1 antibody [EPR22269] (AB227805)

IHC image of vGluT1 staining in a section of frozen normal human cerebellum performed on a Leica BOND^TM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab227805, 0.1ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VGluT1 antibody [EPR22269] (AB227805)

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling VGluT1 with ab227805 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Negative control : No staining on rat liver is observed.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VGluT1 antibody [EPR22269] (AB227805)

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling VGluT1 with ab227805 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Negative control : No staining on mouse liver.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VGluT1 antibody [EPR22269] (AB227805)

ab227805 staining VGluT1 in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab227805 at 1μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VGluT1 antibody [EPR22269] (AB227805)

Immunohistochemical analysis of paraffin-embedded rat cerebral cortex tissue labeling VGluT1 with ab227805 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on rat cerebral cortex (PMID : 29532891) is observed. Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-VGluT1 antibody [EPR22269] (AB227805)

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse hippocampus tissue labeling VGluT1 with ab227805 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Cytoplasmic staining on mouse hippocampus (PMID : 29532891) is observed.

The nuclear counter stain is DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-VGluT1 antibody [EPR22269] (AB227805)

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen rat hippocampus tissue labeling VGluT1 with ab227805 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Cytoplasmic staining on rat hippocampus (PMID : 29532891) is observed.

The nuclear counter stain is DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-VGluT1 antibody [EPR22269] (AB227805)

VGluT1 was immunoprecipitated from 0.35 mg of mouse brain lysate with ab227805 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab227805 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : Mouse brain lysate 10 μg (Input).

Lane 2 : ab227805 IP in mouse brain lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab227805 in mouse brain lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 1 second.

The pattern of oligomeric/dimeric forms observed is consistent with what has been described in the literature (PMID : 15192755).

All lanes:

Immunoprecipitation - Anti-VGluT1 antibody [EPR22269] (ab227805)

Predicted band size: 61 kDa

false

• WB

Supplier Data

Western blot - Anti-VGluT1 antibody [EPR22269] (AB227805)

Blocking/Dilution buffer : 5% NFDM/TBST.

Oligomeric forms observed in the human brain lysate were due to the 1% SDS Hot lysate preparation method, consistent with what has been described in the literature (PMID : 15192755).

All lanes:

Western blot - Anti-VGluT1 antibody [EPR22269] (ab227805) at 1/1000 dilution

All lanes:

Human brain lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 61 kDa

Observed band size: 62 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-VGluT1 antibody [EPR22269] (AB227805)

Blocking/Dilution buffer : 5% NFDM/TBST.

Brain lysates were prepared with RIPA lysis buffer. The pattern of oligomers/dimers observed is consistent with what has been described in the literature (PMID : 15192755).

All lanes:

Western blot - Anti-VGluT1 antibody [EPR22269] (ab227805) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

Rat brain lysate at 20 µg

Secondary

Lane 1:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Lane 2:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 61 kDa

Observed band size: 62 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-VGluT1 antibody [EPR22269] (AB227805)

Blocking/Dilution buffer : 5% NFDM/TBST.

Different lysate preparation and boiling methods resulted in different banding patterns observed. The lysates in lanes 1 and 3 were prepared by RIPA lysis method with and without boiling. The lysate in lane 2 was prepared by 1% SDS Hot lysis method. For WB sample preparation, we recommend using RIPA lysis buffer and without boiling.

All lanes:

Western blot - Anti-VGluT1 antibody [EPR22269] (ab227805) at 1/1000 dilution

All lanes:

Mouse brain lysate at 20 µg/mL

Secondary

Lane 1:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Lanes 2 - 3:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 61 kDa

Observed band size: 62 kDa

false

Exposure time: 3min