A two steps embedding technique for frozen sections

Oral Oncology ◽  
2021 ◽  
Vol 123 ◽  
pp. 105631
Author(s):  
Deepak Pandiar ◽  
Pratibha Ramani ◽  
Reshma Poothakulath Krishnan ◽  
Aklesha Behera ◽  
K. Monica
Keyword(s):  
Frozen Sections ◽  
Embedding Technique

scholarly journals A PARAFFIN EMBEDDING TECHNIQUE FOR STUDIES EMPLOYING IMMUNOFLUORESCENCE

1962 ◽  
Vol 10 (3) ◽  
pp. 250-256 ◽  
Author(s):  
GUY SAINTE-MARIE
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Influenza A Virus ◽  
Bovine Serum ◽  
Influenza A ◽  
Gamma Globulin ◽  
Frozen Sections ◽  
Rabbit Antibody ◽  
Paraffin Embedding ◽  
Embedding Technique

A method is described for the fixation of blocks of tissue for use in studies employing immunofluorescence. This method consists of fixing thin blocks in 95% ethanol and carrying out the subsequent dehydration and clearing at refrigerator temperatures (4°C). Thereafter, embedding in paraffin and sectioning by the standard microtomy is easy. This method results in preparations which are histologically more precise in the localization of antigen or antibody than preparations of frozen tissues; and with rabbit antibody and bovine serum albumin, the sensitivity of detection is enhanced. Bovine serum albumin can be found for longer periods after injection than is possible with frozen sections. Other antigens for which this procedure has proved satisfactory are bovine gamma globulin, horse ferritin, influenza A virus, diphtheria and tetanus toxoids. Hen's ovalbumin deteriorated. New antigens or new antibodies should be tested before being committed to this method.

The 215 KD mannose-6-phosphate receptor is involved in crinophagy but not in autophagy

1990 ◽  
Vol 48 (3) ◽  
pp. 932-933
Author(s):  
Toru Noda ◽  
Mary L. Bronson ◽  
Shang-Ming Yu ◽  
Marilyn G. Farquhar
Keyword(s):  
Lysosomal Enzymes ◽  
Female Rats ◽  
Frozen Sections ◽  
Cell Organelles ◽  
Embedding Technique ◽  
Partial Digestion ◽  
Rough Er ◽  
Mannose 6 Phosphate ◽  
Ultrathin Frozen Sections ◽  
Lysosomal Proteins

Autophagy and crinophagy represent the two major pathways for digestion of intracellular material via lysosomes have been described. Though both phenomena involve in corporation of cell organelles into lysosomes and thus degradation by lysosomal enzymes, the process by which autophagic and crinophagic vacuoles acquire lysosomal enzymes remains to be clarified. The aim of this work is to find out if mannose-6-phosphate (M6P) receptors are involved in this process. As a typical working model, we used hepatocytes of leupeptin-treated rats for autophagy (Fig. 1) and mammotrophs of female rats treated with estradiol for 6 days, for crinophagy. To localize the M6P receptor and lysosomal proteins, we carried out immunocytochemistry, using a immunoperoxidase pre-embedding technique and immunogold labeling on ultrathin frozen sections.Many autophagosomes induced by leupeptin treatment contained recognizable mitochondria and rough ER. The configuration and fine structure of these organelles are gradually lost and the contents of the vacuoles become more homogeneous and electron-dense, suggesting that partial digestion of the contents of autophagosomes still occurs after leupept in treatment.

Immunoferritin localization of intracellular antigens in positively stained frozen sections

1978 ◽  
Vol 36 (2) ◽  
pp. 168-169
Author(s):  
K. T. Tokuyasu
Keyword(s):  
Surface Tension ◽  
Water Surface ◽  
Thin Layers ◽  
The Other ◽  
Positive Staining ◽  
Frozen Sections ◽  
The Past ◽  
Ultrathin Frozen Sections ◽  
Definition Of

During the past investigations of immunoferritin localization of intracellular antigens in ultrathin frozen sections, we found that the degree of negative staining required to delineate u1trastructural details was often too dense for the recognition of ferritin particles. The quality of positive staining of ultrathin frozen sections, on the other hand, has generally been far inferior to that attainable in conventional plastic embedded sections, particularly in the definition of membranes. As we discussed before, a main cause of this difficulty seemed to be the vulnerability of frozen sections to the damaging effects of air-water surface tension at the time of drying of the sections.Indeed, we found that the quality of positive staining is greatly improved when positively stained frozen sections are protected against the effects of surface tension by embedding them in thin layers of mechanically stable materials at the time of drying (unpublished).

Localization of pancreatic enzymes in ultrathin frozen sections stained with metal labeled antibody

1978 ◽  
Vol 36 (2) ◽  
pp. 90-91
Author(s):  
Kenjiro Yasuda
Keyword(s):  
Fluorescent Antibody ◽  
Pancreatic Enzymes ◽  
Tissue Preparation ◽  
Zymogen Granules ◽  
Frozen Sections ◽  
En Bloc ◽  
Antibody Method ◽  
Ultrathin Frozen Sections ◽  
Fluorescent Antibody Method

Localization of amylase,chymotrypsinogen and trypsinogen in pancreas was demonstrated by Yasuda and Coons (1966), by using fluorescent antibody method. These enzymes were naturally found in the zymogen granules. Among them, amylase showed a diffuse localization around the nucleus, in addition to the zymogen granules. Using ferritin antibody method, scattered ferritin granules were also found around the Golgi area (Yasuda et al.,1967). The recent advance in the tissue preparation enables the antigen to be localized in the ultrathin frozen sections, by applying the labeled antibodies onto the sections instead of staining the tissue en bloc.The present study deals with the comparison of the localization of amylase and lipase demonstrated by applying the bismuth-labeled, peroxidase-labeled and ferritin-labeled antibody methods on the ultrathin frozen sections of pancreas, and on the blocks of the same tissue.

Ultrastructural Localization of Acid Mucosubstance in Skeletal Muscle

1967 ◽  
Vol 25 ◽  
pp. 52-53 ◽  
Author(s):  
William J. Dougherty ◽  
Samuel S. Spicer
Keyword(s):  
Striated Muscle ◽  
Divalent Cations ◽  
Ultrastructural Localization ◽  
Major Elements ◽  
Frozen Sections ◽  
Thorium Dioxide ◽  
Iron Solution ◽  
Coupling System ◽  
Psoas Muscles ◽  
Formalin Fixed

In recent years, considerable attention has focused on the morphological nature of the excitation-contraction coupling system of striated muscle. Since the study of Porter and Palade, it has become evident that the sarcoplastic reticulum (SR) and transverse tubules constitute the major elements of this system. The problem still exists, however, of determining the mechamisms by which the signal to interdigitate is presented to the thick and thin myofilaments. This problem appears to center on the movement of Ca++ions between myofilaments and SR. Recently, Philpott and Goldstein reported acid mucosubstance associated with the SR of fish branchial muscle using the colloidal thorium dioxide technique, and suggested that this material may serve to bind or release divalent cations such as Ca++. In the present study, Hale's iron solution adapted to electron microscopy was applied to formalin-fixed myofibrils isolated from glycerol-extracted rabbit psoas muscles and to frozen sections of formalin-fixed rat psoas muscles.

The Application of Ultracryotomy to Immunocytochemistry

1973 ◽  
Vol 31 ◽  
pp. 704-709
Author(s):  
R. G. Painter ◽  
K. T. Tokuyasu ◽  
S. J. Singer
Keyword(s):  
Frozen Sections ◽  
Protein Antigens ◽  
Carbon Coated ◽  
Antibody Conjugates ◽  
Ultrathin Frozen Sections

A technique for localizing intracellular antigens with immunoferritin conjugates directly on ultrathin frozen sections of glutaraldehyde-fixed tissues has been developed. This method overcomes some of the limitations of previously described procedures, since it avoids drastic fixation, dehydration and embedding procedures which could denature many protein antigens.Briefly cells or tissues were fixed with glutaraldehyde (0.5 to 2% for 1 hr), and ultrathin frozen sections were cut and mounted on grids covered with carbon-coated Formvar film by the procedure described previously. Such sections were stained with ferritin-antibody conjugates by methods described elsewhere.

Ultrathin frozen sections of yeast without aldehyde prefixation

1978 ◽  
Vol 36 (2) ◽  
pp. 58-59
Author(s):  
K. J. Böhm ◽  
a. E. Unger
Keyword(s):  
Aqueous Solutions ◽  
Biological Material ◽  
Yeast Cells ◽  
Frozen Sections ◽  
Good Preservation ◽  
Ultrathin Frozen Sections

During the last years it was shown that also by means of cryo-ultra-microtomy a good preservation of substructural details of biological material was possible. However the specimen generally was prefixed in these cases with aldehydes.Preparing ultrathin frozen sections of chemically non-prefixed material commonly was linked up to considerable technical and manual expense and the results were not always satisfying. Furthermore, it seems to be impossible to carry out cytochemical investigations by means of treating sections of unfixed biological material with aqueous solutions.We therefore tried to overcome these difficulties by preparing yeast cells (S. cerevisiae) in the following manner:

Visualization of the Platelet-Plasma Interface

1977 ◽  
Vol 35 ◽  
pp. 494-495
Author(s):  
D. C. Brindley ◽  
M. McGill
Keyword(s):  
Platelet Rich Plasma ◽  
Coagulation Factors ◽  
Platelet Membrane ◽  
Rabbit Platelet ◽  
Surface Coat ◽  
Special Relationship ◽  
Routine Method ◽  
Embedding Technique ◽  
Biochemical Analyses ◽  
Adsorptive Properties

Morphological and cytochemical studies of platelets have reported a surface coat, or glycocalyx, external to the plasma membrane (1). Biochemical analyses have likewise confirmed the highly adsorptive properties of platelets as transporters of coagulation factors (2). However, visualization of the platelet membrane by conventional EM procedures does not reflect this special relationship between the platelet and its plasma environment. By the routine method of alcohol-propylene oxide dehydration for Epon embedding, the lipid bilayer nature of the platelet membrane appears similar to other blood cells (Fig. 1). A new rapid embedding technique using dimethoxypropane (DMP) as dehydrating agent (13) has permitted ultrastructural analyses of the surface features of the platelet-plasma interface.Aliquots of human or rabbit platelet-rich plasma (PRP) were added to equal volumes of 6% glutaraldehyde in Millonig's buffer at 37° for 45 minutes, rinsed in buffer and postfixed in 1% osmium in Millonig's buffer for 45 minutes.

X-Ray Analysis of Frozen Hydrated Sections:The Unconventional Approach

1982 ◽  
Vol 40 ◽  
pp. 754-757
Author(s):  
R. Beeuwkes ◽  
A. Saubermann ◽  
P. Echlin ◽  
S. Churchill
Keyword(s):  
Ratio Method ◽  
Frozen Sections ◽  
Technical Problems ◽  
Sample Handling ◽  
X Ray ◽  
Common Group ◽  
Ideal Method ◽  
Classic Paper ◽  
Physical Principles ◽  
The Ideal

Fifteen years ago, Hall described clearly the advantages of the thin section approach to biological x-ray microanalysis, and described clearly the ratio method for quantitive analysis in such preparations. In this now classic paper, he also made it clear that the ideal method of sample preparation would involve only freezing and sectioning at low temperature. Subsequently, Hall and his coworkers, as well as others, have applied themselves to the task of direct x-ray microanalysis of frozen sections. To achieve this goal, different methodological approachs have been developed as different groups sought solutions to a common group of technical problems. This report describes some of these problems and indicates the specific approaches and procedures developed by our group in order to overcome them. We acknowledge that the techniques evolved by our group are quite different from earlier approaches to cryomicrotomy and sample handling, hence the title of our paper. However, such departures from tradition have been based upon our attempt to apply basic physical principles to the processes involved. We feel we have demonstrated that such a break with tradition has valuable consequences.

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