The periodic acid-schiff reaction in the adrenal medulla

Summary The PAS reaction in the adrenal medulla of rat, rabbit, hamster, ox, pig and sheep was investigated. The medullary cells were positive in cryostat sections and potassium dichromate fixed material but not in formaldehyde fixed paraffin sections. The latter result is due mostly to the extraction of PAS positive lipids and loss of PAS positive proteins. No glycogen was detected in the chromaffin cells histochemically. The catechol amines played no part in the PAS reaction unless the fixative contained dichromate. The connective tissue elements were also PAS positive, and the nerve fibres in ox, sheep and pig. Periodate cannot be used to differentiate between adrenaline and noradrenaline storing cells.     

On the cytochemical demonstration of glycogen in neutrophil granulocytes: periodic acid-Schiff reaction and diastase (amylase) digestion test (author's transl)]

The results of the present investigation indicate clearly that treatment of blood smears with diastase resp. amylase is unsuitable to identify glycogen in neutrophil granulocytes. This may be attributed to the contamination with proteases of commonly used preparations of diastase resp. amylase. Thus strong PAS-reactive material which presents most probably not glycogen but PAS-positive glycoproteins may be eliminated by the proteolytic activity of the contaminants. - In detail it has been shown that susceptibility resp. resistance of the PAS-positive material against treatment with diastase resp. amylase is highly dependent on both type of fixation and fixation time: Fixation with formol free absolute alcohol (ethanol, methanol), leads also after prolonged fixation time to a complete loss of PAS-staining after preliminary treatment with diastase resp. amylase. On the other side after fixation with formol containing fixatives (for example formol/ethanol and acetic acid/formol/ethanol) only after short term fixation practically a complete loss of PAS-staining material is observed. However, after long term fixation more or less complete resistance of the PAS-stainable material against treatment with diastase resp. amylase has been found.     

The Feulgen reaction after glutaraldehyde fixation.

A technique is described for performing the Feulgen reaction for DNA on cells and tissues fixed in glutaraldehyde. Blockade free aldehydes by reducing them with fresh 0.5% NaBH4 in 1% NaH2PO4 for 1 hr at room temperature, then rinse in water. Follow by a Feulgen reaction (hydrolysis at room temperature in 6 N HCl for 20 min, Schiff's reagent for 60 min.). Controls assure the completeness and irreversibility of the borohydride blockade. Cytophotometry shows that the DNA content per nucleus is unaffected by the blockade procedure.     

The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation

The effect of three proteases--trypsin, pepsin, and pronase--on the immunohistochemical staining of keratins with a broad-spectrum monoclonal antibody was investigated in paraffin sections of formalin and ethanol-fixed tissues by means of the peroxidase-antiperoxidase method. Both the length of exposure to the fixative and the duration of proteolysis were varied over a wide range. Ethanol-fixed tissues showed excellent preservation of the antigenicity of keratins, and no appreciable differences in immunostaining related to the length of fixation were found. The use of proteolytic enzymes did not improve these results; on the contrary, it caused rapid tissue disintegration. Formalin-fixed epithelial tissues stained weakly or failed to stain unless they were treated with a proteolytic enzyme. The optimal length of proteolysis varied with the degree of fixation; tissues that were fixed for long periods of time in formalin required longer exposure to a proteolytic enzyme and were more resistant to digestion than were tissues that were fixed briefly. No significant advantage of one protease over another was found in this study. We conclude that a proteolytic step must precede immunostaining for keratins if the tissue is fixed in formalin, but that the digestion period must be adjusted according to the length of exposure to the fixative. The superiority of alcohol over formalin fixation for the preservation of the antigenicity of keratins is confirmed by this study.     

The reaction of formaldehyde with unsaturated fatty acids during histological fixation

Synopsis Formaldehyde reacts with unsaturated fatty acids in tissues during histological fixation. The reaction of formaldehyde with oleic acid has been found to given rise to compounds (adducts) with the following structures: Two other compounds were isolated but their nature is still open to doubt.The equilibrium constant for the initial part of the reaction has been approximately estimated as 0·042 at a room temperature of 22°C. The endothermic heat of reaction has been estimated as approximately 12·6 kcal.The occurrence of these adducts in tissues explains why it is that less lipid can be demonstrated histologically in material that has been stored in form-aldehyde for a considerable length of time.     

Immunocytochemical localization of amylase in gerbil salivary gland acinar cells processed by rapid freezing and freeze-substitution fixation

We examined the immunocytochemical localization of amylase in cryofixed serous acinar cells of gerbil major salivary glands by indirect immunostaining, using anti-gerbil parotid amylase antibody and protein A-gold complex. Fresh tissue blocks were quickly frozen by the metal-contact method, using liquid helium, and were freeze-substituted with either osmium-acetone solution or glutaraldehyde-containing acetone. They were then embedded in an epoxy resin mixture which was polymerized at 60 degrees C. Some tissue blocks substituted with aldehyde-acetone solution were embedded in Lowicryl K4M, polymerized at -30 degrees C. Thin sections of epoxy resin-embedded materials were treated with an oxidizing agent before immunostaining. The labeling density on the materials processed by various protocols for preparatory procedures was quantitatively compared to examine the usefulness of application of cryofixation to immunocytochemistry. The central dense core of heterogeneous secretory granules in the serous acinar cells of the parotid and sublingual glands was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The immunolabeling pattern clearly distinguished between the dense core and the surrounding matrix. Labeling density in the cryofixed materials was about 1.5 times greater than in those processed by conventional chemical fixation. Seromucous secretory granules in the submandibular gland acinar cells were only faintly labeled. The results obtained indicate that application of immunostaining to quick-frozen, substitution-fixed tissues is useful for high-resolution immunocytochemistry.     

Physiological conditions in the midgut in relation to starch digestion and the salivary amylase of the bug Lygus disponsi

Some physiological conditions in the midgut in relation to starch digestion, the nature of the salivary amylase, and the digestion of soluble starch in the midgut were investigated in Lygus disponsi in order to provide evidence for the supposition that Cl⁻, NO₃⁻, and glutamine have a considerable significance for the digestion of soluble starch in the midgut. The activating effect of Cl⁻ and NO₃⁻ on the salivary amylase manifested itself during the whole reaction period and was generally more striking in the earlier stage of the period. It remained still considerable at 20∼30°C, the optimum temperature range for the growth of the bug. The pH-value of midgut content decreased slightly from the first to the third part of midgut, being 5·0 on average, and was appreciably affected by the diet having a large buffer capacity. Even when the bug had fed on plant sap it increased from 5·0 to 5·8. In the midgut of the bug fed with food containing no Cl⁻, no or little, if any, Cl⁻ was detected, which indicates either that this ion was actually absent or that its concentration was diluted largely by the food sap taken in the midgut. It was confirmed that Cl⁻ could be taken in the midgut from the food containing Cl⁻. Starch digestion began already in the first midgut and was usually completed in the second. The digestion of soluble starch in the midgut was accelerated somewhat in the presence of NaCl and KNO₃. On the basis of these facts, it was inferred that, in vivo, Cl⁻, NO₃⁻, and glutamine, especially NO₃⁻, might play an important rôle in the soluble-starch digestion of this bug.