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.

All lanes : Anti-VGluT1 antibody [EPR22269] (ab227805) at 1/1000 dilution

All lanes : Mouse brain lysate

Lysates/proteins at 20 µg/ml per lane.

Secondary
Lane 1 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lanes 2-3 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 62 kDa
Observed band size: 62 kDa


Exposure time: 3 minutes

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.

 

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.

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.

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.

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.

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.

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).

Anti-VGluT1 antibody [EPR22269] (ab227805) at 1/1000 dilution + Human brain lysate at 20 µg

Secondary
VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 62 kDa
Observed band size: 62 kDa


Exposure time: 3 minutes

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

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 2 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 62 kDa
Observed band size: 62 kDa


Exposure time: 3 minutes

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).

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.

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.

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.