重组Anti-VAChT抗体[EPR29154-71] (ab317452)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Rat hippocampus tissue staining TRIM46 with ab307967 at a 1:5000 (0.102 ug/ml) dilution, VAChT with ab317452 at 1:1000 (0.508 ug/ml) dilution and Serotonin transporter with ab308443 at a 1:500 (0.052 ug/ml) dilution followed by secondary antibody Opal Polymer HRP Ms + Rb.

Panel A: merged staining of anti-TRIM46 (green; Opal^™520), anti-VAChT (magenta; Opal^™690) and anti-Serotonin transporter (yellow; Opal^™570) on rat hippocampus.
Panel B: anti-TRIM46 staining the proximal part of the axon in rat hippocampus.
Panel C: anti-VAChT staining vesicle in rat hippocampus.
Panel D: anti-Serotonin transporter staining dendrites in rat hippocampus.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining: in the order of ab307967, ab317452 and ab308443 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal^™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Rat cerebrum tissue staining TRIM46 with ab307967 at a 1:5000 (0.102 ug/ml) dilution, VAChT with ab317452 at 1:1000 (0.508 ug/ml) dilution and Serotonin transporter with ab308443 at a 1:500 (0.052 ug/ml) dilution followed by secondary antibody Opal Polymer HRP Ms + Rb.

Panel A: merged staining of anti-TRIM46 (green; Opal^™520), anti-VAChT (magenta; Opal^™690) and anti-Serotonin transporter (yellow; Opal^™570) on rat cerebrum.
Panel B: anti-TRIM46 staining the proximal part of the axon in rat cerebrum.
Panel C: anti-VAChT staining vesicle in rat cerebrum.
Panel D: anti-Serotonin transporter staining dendrites in rat cerebrum.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining: in the order of ab307967, ab317452 and ab308443 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal^™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse hippocampus tissue staining TRIM46 with ab307967 at a 1:5000 (0.102 ug/ml) dilution, VAChT with ab317452 at 1:1000 (0.508 ug/ml) dilution and Serotonin transporter with ab308443 at a 1:500 (0.052 ug/ml) dilution followed by secondary antibody Opal Polymer HRP Ms + Rb.

Panel A: merged staining of anti-TRIM46 (green; Opal^™520), anti-VAChT (magenta; Opal^™690) and anti-Serotonin transporter (yellow; Opal^™570) on mouse hippocampus.
Panel B: anti-TRIM46 staining the proximal part of the axon in mouse hippocampus.
Panel C: anti-VAChT staining vesicle in mouse hippocampus.
Panel D: anti-Serotonin transporter staining dendrites in mouse hippocampus.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining: in the order of ab307967, ab317452 and ab308443 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal^™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse cerebrum tissue staining TRIM46 with ab307967 at a 1:5000 (0.102 ug/ml) dilution, VAChT with ab317452 at 1:1000 (0.508 ug/ml) dilution and Serotonin transporter with ab308443 at a 1:500 (0.052 ug/ml) dilution followed by secondary antibody Opal Polymer HRP Ms + Rb.

Panel A: merged staining of anti-TRIM46 (green; Opal^™520), anti-VAChT (magenta; Opal^™690) and anti-Serotonin transporter (yellow; Opal^™570) on mouse cerebrum.
Panel B: anti-TRIM46 staining the proximal part of the axon in mouse cerebrum.
Panel C: anti-VAChT staining vesicle in mouse cerebrum.
Panel D: anti-Serotonin transporter staining dendrites in mouse cerebrum.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining: in the order of ab307967, ab317452 and ab308443 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal^™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

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

Low expression: liver, kidney (PMID: 9427309).

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 22087075, PMID: 7559575, PMID: 34230437).

The identity of the bands higher than 100 kDa (in lanes 1-4) are unknown.

The identity of the lower MW band at approximately 37 kDa (in lane 4) is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

Exposure time: Lane 2: 26 seconds, lanes 1, 3-5: 114 seconds

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

VAChT is a glycoprotein of approximately 75 kDa and detected as a 60-kDa band after treated with Peptide:N-glycosidase F (PNGase F).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

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

Low expression: liver, cerebellum (PMID: 9427309).

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 22087075, PMID: 7559575, PMID: 34230437).

The identity of the bands higher than 100 kDa and lower than 50 kDa are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling VAChT with ab317452 at 1/1000 (0.508 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse cerebrum. The section was incubated with ab317452 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling VAChT with ab317452 at 1/1000 (0.508 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat cerebrum. The section was incubated with ab317452 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling VAChT with ab317452 at 1/1000 (0.508 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression:: no staining on mouse liver. The section was incubated with ab317452 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling VAChT with ab317452 at 1/1000 (0.508 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression:: no staining on rat liver. The section was incubated with ab317452 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum (fresh frozen) tissue labeling VAChT with ab317452 at 1/100 (5.08 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Panel A: merged staining of anti-VAChT (ab317452, green), anti-NeuN (ab190565, magenta) on mouse cerebrum.
Panel B: anti-VAChT stained on mouse cerebrum.
Panel C: anti-NeuN stained in neurons of mouse cerebrum.
The section was incubated in two rounds of staining: in the order of ab317452 and ab190565 for 1 hr at room temperature. The nuclear counterstain was DAPI (Blue). The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 (2 ug/mL) dilution.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse liver (fresh frozen) tissue labeling VAChT with ab317452 at 1/100 (5.08 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Low expression: confocal image showing no staining on mouse liver (PMID: 9427309). The nuclear counterstain was DAPI (Blue). The section was incubated with ab317452 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 (2 ug/mL) dilution.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebrum (fresh frozen) tissue labeling VAChT with ab317452 at 1/100 (5.08 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Panel A: merged staining of anti-VAChT (ab317452, green), anti-NeuN (ab190565, magenta) on rat cerebrum.
Panel B: anti-VAChT stained on rat cerebrum.
Panel C: anti-NeuN stained in neurons of rat cerebrum.
The section was incubated in two rounds of staining: in the order of ab317452 and ab190565 for 1 hr at room temperature. The nuclear counterstain was DAPI (Blue). The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 (2 ug/mL) dilution.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat liver (fresh frozen) tissue labeling VAChT with ab317452 at 1/100 (5.08 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Low expression: confocal image showing no staining on rat liver (PMID: 9427309). The nuclear counterstain was DAPI (Blue). The section was incubated with ab317452 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 (2 ug/mL) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse hippocampal neuron cells labelling VAChT with ab317452 at 1/100 (5.08 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining in mouse hippocampal neuron (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain at 1/500 (4ug/ml) dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized mouse primary neuron cells labelling VAChT with ab317452 at 1/500 dilution (0.1 ug)/Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control.

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

VAChT was immunoprecipitated from 0.35 mg Mouse brain tissue lysate with ab317452 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317452 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain tissue lysate
Lane 2: ab317452 IP in Mouse brain tissue lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab317452 in mouse brain whole cell lysate

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

Exposure time: 58 seconds.