Sections Of Aldehyde-fixed Tissue Research Articles

The use of metal-catalyzed oxidation to suppress background staining caused by marker amplification reagents and the effects of this oxidation on the stability of antibody-antigen complexes in immunohistochemistry

ABSTRACTMarker amplification is a powerful technique for visualizing immunohistochemically deposited markers that otherwise would be invisible. Amplification usually is achieved with physical developers, which are solutions that contain a source of silver(I) plus a reducing agent. When the marker is present in extremely small quantities, prolonged incubation in the developer is required and unwanted background staining in the form of type III argyrophilia becomes problematic. Suppression of type III argyrophilia can be achieved by metal-catalyzed oxidation using the copper/H2O2 system, which normally is applied immediately prior to amplification. Because there is no reason, in principle, why metal-catalyzed oxidation should not be employed at earlier stages in the immunohistochemical staining procedure, we investigated whether earlier oxidation might confer any advantages over the traditional methodology. Immunocolloidal gold combined with two light insensitive physical developers was chosen as the model system, because visualization by light microscopy requires extended periods in the developers. Moreover, the system does not suffer from problems concerning endogenous enzyme- or non-enzyme-catalyzed marker deposition. Applying metal-catalyzed oxidation at each stage of the immunohistochemical procedure revealed that the technique could be employed successfully prior to staining, but not following the primary or secondary antibodies. In the latter cases, specific immunolocalization was lost entirely and only generalized nonspecific staining was seen. A limited investigation into the mechanism of metal-catalyzed oxidation of aldehyde fixed tissue sections suggested that it involved the formation of aldehyde groups. We suggest that the application of metal-catalyzed oxidation prior to immunohistochemical staining would have the advantages of both suppressing type III argyrophilia and inhibiting unwanted endogenous peroxidase activity. We also suggest that metal-catalyzed oxidation might reduce the affinity of tissue for other transition metals, such as copper, whose potential for improving marker amplification techniques has been demonstrated previously in dot-blot model systems.

A modified semipermeable membrane technique for carbonic anhydrase histochemistry of the nervous system.

We present a modification of Hansson's method for the demonstration of carbonic anhydrase activity. Using a semipermeable membrane together with a fluid incubation medium, frozen sections of aldehyde-fixed tissue were incubated without floating or dipping. Thin sections (thickness, 20-40 microns) were mounted on the outer surface of a tubular-shaped, semipermeable cellophane dialysis membrane containing the incubation fluid. After incubation for 25-30 min at room temperature, the sections were rinsed in buffer and treated with 0.5% (NH4)2S solution. The histochemical reaction was fully inhibited by 10(-4) M acetazolamide.

Ultrahistochemical reactions of the suberized cell walls in Acorus, Acacia, and Larix

Summary Suberized cell walls of Acorus and Acacia show a non-periodate-reactive suberinic layer between the strongly reactive primary and tertiary wall layers. Structure and reactivity of the tertiary and primary walls are alike and differ from the weaker reacting secondary walls of other cells. This type of suberized cell wall contrasts with that of Larix and Picea where the wall formation ends with the deposition of the non-periodate-reactive filling cork. Lamellae in the suberinic layer cannot only be contrasted by block fixation with osmium, but also by treating thin sections of aldehyde-fixed tissue with osmium, showing that no Liesegang processes are implicated in the lamella formation. A granular silver contrast arranged in lamellae can be provoked in the suberinic layer by a treatment with sulphuric acid and iodine, followed by silver proteinate. The acid concentration must be higher for Larix than for Acacia. This reaction seems to correspond to a violet stain of the suberinic layer with zinc-chlor-iodide, shown by Gilson (1890) to be specific for phellonic acid.