Reappraisal of potassium permanganate oxidation applied to Lowicryl K4M embedded tissues processed by high pressure freezing/freeze substitution, with special reference to differential staining of the zymogen granules of rat gastric chief cells.
The high pressure freezing/freeze substitution technique is known to yield a deep vitreous freezing of tissues. Combination of this technique with Lowicryl K4M embedding allows us histochemical studies of dynamic cellular processes with improved structural preservation. The disadvantage of Lowicryl K4M embedding is its poor electron density in electron microscopy. To address this problem, we examined the effects of KMnO4 oxidation applied to Lowicryl K4M embedded rat gastric glands processed by high pressure freezing. The KMnO4 oxidation-uranyl acetate-lead citrate sequence succeeded not only in contrast enhancement of cellular components, but also in differential staining of the zymogen granules of rat gastric chief cells. This technique could be applied to semi-thin sections of Lowicryl K4M embedded rat gastric glands. The KMnO4 oxidation-toluidine blue staining provided sufficient contrast with regard to the zymogen granules. Various experiments used in this study verified that the KMnO4 oxidation plays an essential role in the differential staining of the zymogen granules. Combined use of the KMnO4 oxidation with phospholipase A2-immunostaining demonstrated that gold labeling was localized to the zymogen granules without the loss of immunolabeling. Energy dispersive X-ray microanalysis revealed some manganese depositions on the zymogen granules. It is highly anticipated that the KMnO4 oxidation will become a useful tool for histochemical investigations combined with cryofixation/freeze substitution and low temperature embedding techniques.
Sawaguchi
A
A
Department of Anatomy, Miyazaki Medical College, Japan.
Ide
S
S
Kawano
J
J
Nagaike
R
R
Oinuma
T
T
Tojo
H
H
Okamoto
M
M
Suganuma
T
T
D000180:Acrylic Resins; D000818:Animals; D019872:Chief Cells, Gastric / Q000201:enzymology* / Q000648:ultrastructure*; D002951:Citrates; D004396:Coloring Agents; D003594:Cytoplasmic Granules / Q000201:enzymology / Q000648:ultrastructure; D004577:Electron Probe Microanalysis; D004792:Enzyme Precursors / Q000032:analysis*; D005404:Fixatives; D017110:Freeze Substitution / Q000379:methods*; D007854:Lead; D008297:Male; D008854:Microscopy, Electron / Q000379:methods; D009942:Organometallic Compounds; D010084:Oxidation-Reduction; D010741:Phospholipases A / Q000032:analysis; D054467:Phospholipases A2; D011196:Potassium Permanganate / Q000378:metabolism* / Q000494:pharmacology; D011312:Pressure; D051381:Rats; D017208:Rats, Wistar; D013194:Staining and Labeling / Q000379:methods; D016610:Tissue Embedding / Q000379:methods
D000180:Acrylic Resins; D002951:Citrates; D004396:Coloring Agents; D004792:Enzyme Precursors; D005404:Fixatives; D009942:Organometallic Compounds; D011196:Potassium Permanganate; C005460:uranyl acetate; D007854:Lead; C037883:Lowicryl 4KM resin; D010741:Phospholipases A; D054467:Phospholipases A2