Blocking solution

Tissue prints on nitrocellulose membranes (Schleicher Schuell) (0.45 pm pore size) were first stained for protein using Ponceau S (Fig. 2A,B) and photographed immediately with T-max 100 black-and-white film. The membranes were then washed in PBS to remove the stain, and incubated for 2 h with shaking in blocking solution. 

After blocking, the initial step for immunolabeling is to incubate grids in blocking solution in which is diluted the primary antibody. Grids can either be floated on the solution or immersed within in it. We use... 

Treat grid with blocking solution (1% bovine serum albumin [BSA], 3% gelatin, 0.9% NaCl in 0.01 M phosphate buffer, pH 7.4). 

Primary antibodies blot excess blocking solution from sections, and incubate for 60 min at room temperature or over night at +4°C with a correspondingly diluted primary antibody. Wash sections in PBS or TBS for 2 x 3 min. 

Blocking step incubate sections for 20 min with normal serum blocking solution (see Sect. 5.1). 

Normal Serum Blocking Solution 2 5% normal serum (blocking)... 

Primary antibodies blot excess blocking solution from sections and incubate for 60 min at room temperature or overnight at +4°C with a mixture of correspondingly diluted unlabeled primary antibodies raised in two different host species (e.g., mouse and rabbit). When using fluorophore-labeled primary antibodies as in direct immunostaining method (one antibody layer), you may skip step (6) with secondary antibodies for indirect immunostaining method (two antibodies layers). Wash sections in PBS for 3 x 3 min. 

Ready-to-use blocking solutions containing normal inactivated sera or BSA for use with colloidal gold conjugates are available from Aurion (http //www. aurion.nl/). When labeling with protein A and protein G conjugates, this step can be omitted. 

PIPAAm-PBMA block copolymers form a micellar structures by selfassociation of the hydrophobic PBMA segments in water, a good solvent for PlPAAm chains below the LCST but a nonsolvent for the PBMA chains. This amphiphilic system produces stable and monodispersed micelles from polymer/A-ethylacetamide (good solvent for the both polymer blocks) solutions dialyzed against water. Hydrophobic dmgs can be physically incorporated into the iimer micelle cores with PBMA chains by hydrophobic interactions between the hydrophobic segments and dmgs. 

Blocking solution 5g of fat free milk in 100 ml of TBST. (Carnation milk works well)... 

After blocking, incubate the blot with primary antibody at an appropriate dilution in 10 ml of blocking solution. 

Add 15 ml of blocking solution with appropriate amount of secondary antibody (Either Alkaline phosphatase or HRP conjugated antibody). 

Endogenous enzyme blocking solution, 1.5% hydrogen peroxide (H2O2) in PBS prepared from a dilution of 30% H2O2. 

Serum blocking solution, 10% animal serum in PBS (the species of serum should match the detecting system antibody, see Note 1). 

Block endogenous peroxidase and peroxidase-like activity by incubation in 1.5% H2O2 in PBS endogenous enzyme blocking solution for 15 min with constant stirring (see Note 8). 

Block charged sites on tissue surface with incubation in the 10% serum in PBS serum blocking solution overnight at 4°C (see Note 1). 

The enzyme or the chromogen detection system determines whether any endogenous material must first be destroyed. If a peroxidase marker molecule is to be used, endogenous peroxidase or peroxidase-like activity should be blocked. Because these preparations are more fragile than a fixed embedded sample, endogenous enzyme is inactivated with a weaker blocking solution than would... 

Nonspecific binding blocking solution 10% normal serum of the secondary antibody generating species in PBS. 

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