Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Immunohistochemical analysis of formalin fixed paraffin embedded human brain (cerebrum) labelling Olig2 with ab109186 at a concentration of 0.96 µg/ml. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins. ab109186 Anti-Olig2 antibody [EPR2673] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Immunohistochemical analysis of formalin fixed paraffin embedded human brain (cerebrum) labelling Olig2 with ab109186 at a concentration of 0.96 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32 mins at 100°C with ULTRA cell conditioning solution (CC1, pH 8.5). ab109186 Anti-Olig2 antibody [EPR2673] was incubated at 37°C for 16 mins. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

ab109186 staining Olig2 in primary mouse neurons/glia, DIV14 (prepared from E18 mouse hippocampal brain area, obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. C57EHP) cells. 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 ab109186 at 5µg/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). Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cortex (fresh frozen) tissue labeling Olig2 with ab109186 at 1/100 (10 μg/mL) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Confocal image showing positive staining on mouse cortex. The section was incubated with the primary antibody for 60 mins 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) preadsorbed at 1/1000 (2 ug/mL) dilution.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cortex (fresh frozen) tissue labeling Olig2 with ab109186 at 1/100 (10 μg/mL) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Confocal image showing positive staining on rat cortex. The section was incubated with the primary antibody for 60 mins 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) preadsorbed at 1/1000 (2 ug/mL) dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse cerebrum tissue labelling NeuN with ab177487 at 1/100 dilution (B), SOX1 with ab242125 at 1/100 dilution (C) and Olig2 with ab109186 at 1/100 dilution (D). Anti-Rabbit and Mouse Polymer HRP was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins. Heat mediated antigen retrieval (Leica ER2, PH9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibodies from the previous round, to avoid any cross-reactivity.

Panel A : merged staining of anti- NeuN (green, Opal™520), anti-SOX1 (red, Opal™570) and anti- Olig2 (yellow, Opal™690).

Panel B : anti-NeuN stained for neurons.

Panel C : anti-SOX1 stained on neural progenitors.

Panel D : anti-Olig2 stained on oligodendrocyte.

The section was incubated in three rounds of staining : in the order of ab177487, ab242125 and ab109186 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

• WB

Unknown

Western blot - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (ab109186) at 1/2000 dilution

All lanes:

Human fetal brain tissue lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 19 kDa,32 kDa,34 kDa,36 kDa,41 kDa,55 kDa

Observed band size: 32 kDa,53 kDa,55 kDa

false

• WB

Unknown

Western blot - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (ab109186) at 1/10000 dilution

All lanes:

Human oligodendroglioma lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 32 kDa

Observed band size: 32 kDa

false

• WB

Unknown

Western blot - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (ab109186) at 1/2000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

Rat brain lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 11 kDa,16 kDa,25 kDa,27 kDa,28 kDa,32 kDa,41 kDa,52 kDa,59 kDa,83 kDa

Observed band size: 25 kDa,30 kDa,32 kDa

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Immunohistochemical staining of Olig2 in human glioma tissue with ab109186 at a dilution of 1/100.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Immunohistochemical staining of paraffin embedded rat cerebral cortex with purified ab109186 at a working dilution of 1/100. The secondary antibody used is ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

ab109186 staining Olig2 in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 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 ab109186 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 100% methanol (5 min).

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

• IMC

Collaborator

Imaging Mass Cytometry - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Imaging Mass Cytometry™ (IMC™) image of human glioblastoma brain cancer tissue stained with Anti-Olig2 antibody [EPR2673]. ab220796 (carrier-free antibody, purified) was metal-conjugated using a Maxpar^® Antibody Labeling Kit from Fluidigm. Immunostaining was performed according to Fluidigm's protocols. Briefly, slides were subject to deparaffinization and heat-induced epitope retrieval, followed by overnight incubation at 4°C with an antibody cocktail containing metal-tagged antibodies in blocking buffer. Slides were subsequently washed with 0.2% Triton-X and 1x PBS, counterstained with Cell-ID™ Intercalator-Ir diluted at 1/400 in 1x PBS for 30 min at room temperature, rinsed for 5 min with distilled H2O, and air-dried prior to IMC™ acquisition. IMC™ acquisition was performed using the Fluidigm Hyperion™ Imaging System.

Imaging Mass Cytometry™, IMC™, Cell-ID™, Hyperion™ and Maxpar^® are trademarks of Fluidigm Canada.

This image is courtesy of the Single Cell & Imaging Mass Cytometry Analysis Platform, Goodman Cancer Research Centre, McGill University

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized rat primary glia cell cells labelling Olig2 with ab109186 at 1/100 (1.23 μg/mL) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (2 μg/mL) (Green). Confocal image showing nuclear staining in rat primary glia cell. Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 μg/mL) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (2 μg/mL).

• IHC-P

AbReview62322****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Formaldehyde-fixed mouse brain tissue stained for Olig2 using ab109186 at 1/100 dilution in immunohistochemical analysis. The secondary antibody was a Horse Radish Peroxidase conjugated Dako Envision Rabbit antibody.

Antigen retrieval : Heat mediated - Buffer/Enzyme Used : pH 9.0 EDTA

This image was courtesy of an annoymous Abreview

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Immunohistochemical staining of paraffin embedded human cerebral cortex with purified ab109186 at a working dilution of 1/100. The secondary antibody used is ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• WB

Lab

Western blot - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

Different batches of ab109186 were tested on Mouse brain lysate at 0.1 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 32 kDa.

All lanes:

Western blot - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (ab109186)

Predicted band size: 32 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Olig2 antibody [EPR2673] - Oligodendrocyte Marker (AB109186)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.