Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling Neurocan with ab277525 at 1/4000 (0.133 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human cerebrum (PMID: 9795216). The section was incubated with ab277525 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

All lanes : Anti-Neurocan antibody [EPR23425-68] (ab277525) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse brain tissue lysate treated with 0.05U chondroitinase ABC for 3h at 37?.
Lane 3 : Rat brain tissue lysate
Lane 4 : Rat brain tissue lysate treated with 0.05U chondroitinase ABC for 3h at 37?.

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 143 kDa
Observed band size: 150-300 kDa why is the actual band size different from the predicted?

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

Neurocan proteolytic cleavage results in two fragments: neurocan-130 (130 kDa) and neurocan-C (150k Da ). Neurocan is a glycoprotein and seen as broad indistinct smears, which on chondroitinase treatment resolve into band of  150 (neurocan-C). The molecular weight observed is consistent with what has been described in the literature (PMID: 10729323).

Exposure time: 48 seconds.

 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neuron cells labelling Neurocan with ab277525 at 1/50 (10.6 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in mouse primary neuron. Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection is observed. ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain MAP2 at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

-ve control 1: ab277525 at 1/50 dilution, followed by ab150120 at 1/1000 dilution.

-ve control 2: ab11267 at 1/200 dilution, followed by ab150077 at 1/1000 dilution.

All lanes : Anti-Neurocan antibody [EPR23425-68] (ab277525) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse hippocampus tissue lysate
Lane 3 : Mouse skeletal muscle tissue lysate

Lysates/proteins at 40 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 143 kDa
Observed band size: 130-300 kDa why is the actual band size different from the predicted?

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

Neurocan is a glycoprotein. The observed MW is consistent with what has been described in the literature (PMID: 29670169).

Negative control: mouse skeletal muscle (PMID: 1326557)

Samples are non-boiled as boiling may cause protein aggregates.

Exposure time: 26 seconds.

 

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling Neurocan with ab277525 at 1/4000 (0.133 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse cerebrum (PMID: 30356641) . The section was incubated with ab277525 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

All lanes : Anti-Neurocan antibody [EPR23425-68] (ab277525) at 1/1000 dilution

Lane 1 : Rat brain tissue lysate
Lane 2 : Rat hippocampus tissue lysate
Lane 3 : Rat skeletal muscle tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 143 kDa
Observed band size: 130-300 kDa why is the actual band size different from the predicted?

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

Neurocan is a glycoprotein. The observed MW is consistent with what has been described in the literature (PMID: 29670169). 

Negative control: rat skeletal muscle (PMID: 1326557).

Samples are non-boiled as boiling may cause protein aggregates.

This blot was developed using a higher sensitivity ECL substrate.

Exposure time: 103 seconds.

 

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling Neurocan with ab277525 at 1/4000 (0.133 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat cerebrum. The section was incubated with ab277525 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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