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.

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 stands 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

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).

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

All lanes : Anti-Olig2 antibody [EPR2673] (ab109186) at 1/2000 dilution (purified)

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

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 32 kDa
Observed band size: 32 kDa

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

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.

Equilibrium disassociation constant (K_D)
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Anti-Olig2 antibody [EPR2673] (ab109186) at 1/10000 dilution (purified) + Human oligodendroglioma lysate at 10 µg

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

Predicted band size: 32 kDa
Observed band size: 32 kDa

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Anti-Olig2 antibody [EPR2673] (ab109186) at 1/2000 dilution (purified) + Human fetal brain tissue lysate at 20 µg

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

Predicted band size: 32 kDa
Observed band size: 32 kDa

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

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.

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.