IHC image of MHC Class II staining in Rat normal spleen formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab23990, 10µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine, and sodium azide (ab23990).

ab23990 staining MHC Class II in Rat spleen tissue by Immunohistochemistry (Frozen sections). The sections were fixed in PFA prior to blocking with 10% serum for 20 minutes at 24°C. The primary antibody was diluted 1/100 in PBS and incubated with the sample for 16 hours at 4°C. An Alexa Fluor® 488-conjugated donkey anti-mouse polyclonal was used as the secondary antibody, diluted 1/1000 for 1 hour at room temperature. Counterstained with Hoechst 33258 (blue) for 10 minutes at room temperature.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine, and sodium azide (ab23990).

Immunohistochemistical detection of MHC Class II using antibody ab23990 on PFA-fixed rat spleen tissue sections. Antibody diluted at 1/100 and incubated for2 hours in  TBS/BSA/Tween/azide. Secondary antibody: anti mouse IgG conjugated to biotin (1/100). After dissection of spleen from PFA-perfused specimen it was sampled and further immersion-fixed for two Hrs. After subsequent immersion in 30% Sucrose, specimens were snap-frozen. Before immunostaining, the 8 micron sections were placed in a 60 degree C oven for 60 mins to enhance adhesion. The submitted image shows white pulp (PALS) and a small area of red pulp (RP-upper left). The Periarteriolar Sheath (PALS) with it's Central Arteriole/artery (CA)shows many positive cells (B-lymphocytes and Macrophages) and negative lymphocytes (T-cells?). The Marginal Sinus (MS) is clearly seen between the PALS and the Marginal Zone (MZ). There is a clear Germinal Centre (GS) in this image.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine, and sodium azide (ab23990).