Normal goat serum

Post-HIER immunostaining buffer 0.05 M Tris-buffered saline (TBS, pH 7.6) containing 1-2% fetal calf serum (FCS) or normal goat serum. 

Blocking solution 10% normal goat serum in PBS with 0.1% sodium azide as a preservative (see Note 3). 

Blocking solution (BS) TBS with 1% bovine serum albumin (BSA), 0.1% sodium azide, and 5% normal goat serum (NGS). 

Block sections with 1% BSA or 5% normal goat serum in TBS. 

Incubate grids for 60 min with primary antibody diluted with TBS containing 1% BSA or 5% normal goat serum (see Note 4). 

Antibody staining buffer PBS containing 0 5% (v/v) Tween-20 and either 1% (w/v) BSA (for direct antibody conjugates), or 0 5% (v/v) normal goat serum when a second antibody is used. 

The sections are washed in two changes of 5 min each in Tris buffer I (2.5% NaCl and 0.55% Tween 20, diluted in 0.05 mol/liter Tris-HCl buffer, pH 8.2). The excess buffer is removed by wiping around the sections, which are then covered for 10 min with 75 pi of normal goat serum (NGS) diluted 1 1 with PBS (pH 7.4). Excess NGF is removed by wiping around the sections. 

Standard antibody dilution buffers such as PBS with BSA or normal goat serum as blockering agents can be used, provided generous detergent (0.5-1% triton X-100) is also included. 

Immunostaining buffer TBS containing 1% BSA or normal goat serum. 

Quickly transfer the slides to TBS and wash, dipping in and out of the solution ten times. Transfer to another Coplin jar containing TBS for 5 min. Transfer to another Coplin jar for a third wash in TBS plus 1-3% normal goat serum for 5 min (see Notes 11 and 12). 

The rinse buffer solution contains 10 mg/ml BSA and 5.0% normal goat serum in PBS (no detergent) use this for all rinses. Mix only as much as needed for the current experiment. To prepare this solution, multiply the number of samples times the volume of each rinse, times the total number of rinses for the entire experiment. For this example experiment, we have 17 experimental samples and 3 controls samples for 20 total samples. For the rinse buffer, use 500 p.1 for each rinse and a total of 14 rinses (two rinses after block and permeabilize six rinses after 1° antibody six rinses after 2° antibody). A total of 20 samples x 14 total rinses x 0.5 ml per rinse = 140 ml of rinse buffer plus 5% excess (7 ml) is 147 ml. The excess amount is a safety factor, in case of a spill or other unexpected event. 

Use rinse buffer solution called PBS plus contains 10 mg/ml BSA, 5.0% normal goat serum, and 0.02% sodium azide in PBS (no detergent). 

Use rinse buffer solution containing 10 mg/ml BSA and 5.0 % normal goat serum in PBS and rinse six times... 

Permeabilization was done with 0.3% Triton X-1000 in PBS containing 5% normal goat serum, 10 mg/ml of BSA. 

Insufficient or wrong blocking agents - Blocking with 5% normal goat serum the same species as the 2° antibody, 10 mg/ml of BSA were sufficient for other rabbit 1° antibodies. 

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