A new method for producing a specific and high titre antibody against glutamate using colloidal gold as a carrier

Polyethylene glycol (PEG) is an excellent embedding medium for immunohistochemical studies. It provides structural preservation superior to frozen sections and increased sensitivity of antigen detection compared with paraffin sections. One limitation of PEG embedment is that PEG sections are difficult to handle and adhere poorly to glass slides. Here we present a simple and effective method for embedding tissues in PEG and transferring the resultant sections onto silanated glass slides. In addition, a method for silver enhanced colloidal gold immunostaining was combined with common dye staining to demonstrate the excellent structure preservation and sensitive antigen detection. Bovine chorionic membrane was fixed with Bouin's fixative, embedded in polyethylene glycol (PEG) 1500, cut into 5-microns sections, flattened over agarose blocks (10 x 10 x 2 mm3), and blotted onto Digene silanated slides. Slides were then washed in PBS, which removed the PEG and agarose blocks. Tissue sections were immunocytochemically stained with dilute antiserum raised in a rabbit against purified bovine placental retinol binding protein (bpRBP). Sections were washed and incubated with 1-nm colloidal gold-labeled goat anti-rabbit IgG. The immunogold particles were enhanced by silver staining (IGSS). Specimens were observed and photographed with an Olympus epipolarization microscope. The new method offered excellent morphological preservation of cell structure and the epipolarization microscopy provided high sensitivity for detection of specific immunogold-silver particles.

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High-resolution, extended views of membrane surfaces revealed by fracture-flip

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155669 ◽

1989 ◽

Vol 47 ◽

pp. 738-739

Author(s):

Pedro Pinto da Silva

Keyword(s):

High Resolution ◽

Colloidal Gold ◽

Cell Membranes ◽

Freeze Fracture ◽

Surface Structures ◽

New Method ◽

Deep Etching ◽

Membrane Surfaces ◽

Routine Identification ◽

Freeze Etching

I will describe a new method — fracture-flip — that uses commercially available equipment to produce extended views of cell and membrane surfaces. The resolution of this new method permits the routine identification of surface structures down to 5 nm diameter. Moreover, in contrast to freeze-etching/deep-etching, extended views are easily obtained.Conceptally, fracture-flip derives from label-fracture, another method developed in my laboratory. With label-fracture we showed that, after freeze-fracture, the exoplasmic (E) halves of cell membranes are stabilized by, and remain attached to, their platinum/carbon replicas. This allows the observation of co-incident views of the Pt/C replica of the E face, and of the distribution of colloidal gold labeled receptors or antigens. This is the sequence of steps in fracture-flip:

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A NEW METHOD OF PREPARING COLLOIDAL SILVER AND GOLD BY MEANS OF A CONTINUOUS HIGH FREQUENCY ELECTRICAL DISCHARGE

Canadian Journal of Research ◽

10.1139/cjr32-065 ◽

1932 ◽

Vol 7 (2) ◽

pp. 133-136 ◽

Cited By ~ 6

Author(s):

A. N. Fraser ◽

J. Gibbard

Keyword(s):

High Frequency ◽

Colloidal Gold ◽

Electrical Discharge ◽

Silver Oxide ◽

New Method ◽

Colloidal Silver ◽

Protective Colloid ◽

Sufficient Intensity

A new method of preparing metal sols, by the use of a continuous high frequency electrical discharge of sufficient intensity to produce a sinusoidal spark, is described. Stable yellow colloidal silver and purple colloidal gold have been prepared, without a protective colloid, by this method. Chemical determinations have failed to indicate any silver oxide in the yellow silver sols.

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