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

1986 ◽  
Vol 34 (8) ◽  
pp. 1095-1100 ◽  
Author(s):  
H Battifora ◽  
M Kopinski
Keyword(s):  
Monoclonal Antibody ◽  
Immunohistochemical Staining ◽  
Proteolytic Enzyme ◽  
Proteolytic Enzymes ◽  
Paraffin Sections ◽  
Optimal Length ◽  
Protease Digestion ◽  
Wide Range ◽  
Excellent Preservation ◽  
Formalin Fixed

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.

Immunohistochemical Detection of Canine Adenovirus in Paraffin Sections of Liver

1986 ◽  
Vol 23 (4) ◽  
pp. 478-484 ◽  
Author(s):  
P. M. Rakich ◽  
K. W. Prasse ◽  
P. D. Lukert ◽  
L. M. Cornelius
Keyword(s):  
Immunohistochemical Detection ◽  
Hepatic Inflammation ◽  
Paraffin Sections ◽  
Inflammatory Lesions ◽  
Long Term Stability ◽  
Formalin Fixed Paraffin ◽  
Wide Range ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

An avidin-biotin immunoperoxidase procedure was optimized for detection of canine adenoviral antigens in formalin-fixed, paraffin-embedded liver. Long-term stability of viral antigen was shown by successful demonstration of virus in liver tissue preserved up to six years from dogs with infectious canine hepatitis. This immunohistochemical stain was applied to sections from livers with a wide range of inflammatory lesions. Examination of sections from 53 dogs yielded five livers with small amounts of adenovirus. An additional virus-positive liver was identified from a dog with no hepatic inflammation. Although a cause and effect relationship remains to be determined, these findings suggest a possible connection between canine adenovirus and spontaneous chronic hepatitis.

scholarly journals Immunostaining of chain-specific keratins on formalin-fixed, paraffin-embedded tissues: a comparison of various antigen retrieval systems using microwave heating and proteolytic pre-treatments.

1995 ◽  
Vol 43 (4) ◽  
pp. 429-437 ◽  
Author(s):  
H M Hazelbag ◽  
L J van den Broek ◽  
E B van Dorst ◽  
G J Offerhaus ◽  
G J Fleuren ◽  
...  
Keyword(s):  
Microwave Heating ◽  
Proteolytic Enzymes ◽  
Antigen Retrieval ◽  
Detergent Solution ◽  
Immunoperoxidase Method ◽  
Paraffin Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Pre Treatment ◽  
Formalin Fixed

The use of chain-specific monoclonal antibodies (MAbs) against keratins in pathology is hampered by their limited staining on formalin-fixed, paraffin-embedded tissue. In the present study, various treatments before immunohistochemistry on paraffin sections were compared, including proteolytic enzymes and microwave antigen retrieval in various solutions. Sections of normal cervical and skin tissue were stained in a three-step immunoperoxidase method, employing a broad panel of MAbs against chain-specific keratins 4, 5, 7, 8, 10, 13, 14, 17, 18, 19 and pankeratin. Using microwave heating, Target Unmasking Fluid (TUF), Antigen Retrieval Solution (ARS), a simple detergent solution (DET), PBS, and distilled water (MiQ) were compared. Microwave heating in PBS or MiQ strongly improved staining results. Moreover, microwave pre-treatment in TUF or DET gave excellent and specific staining with the majority of MAbs tested, comparable with or even better than staining obtained on frozen sections. Using microwave antigen retrieval, tissue morphology remained optimal, and only in a very limited number of MAbs did immunoreactivity on paraffin sections fail to be restored. Proteolytic pre-treatment with trypsin, pepsin, or pronase gave moderate to strong staining with some of the MAbs. Other MAbs, for which microwave pre-treatment was able to restore the loss of immunoreactivity, failed to give appropriate staining with proteolytic pre-treatment. Our results show that microwave heating in either TUF or a simple detergent solution before immunohistochemistry is a reliable method for antigen retrieval of chain-specific keratins in formalin-fixed, paraffin-embedded tissues.

scholarly journals Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

2021 ◽  
Vol 22 (15) ◽  
pp. 7906
Author(s):  
Alexey A. Komissarov ◽  
Maria A. Karaseva ◽  
Marina P. Roschina ◽  
Andrey V. Shubin ◽  
Nataliya A. Lunina ◽  
...  
Keyword(s):  
Cell Death ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Proteolytic Enzymes ◽  
Morphological Changes ◽  
Human Cells ◽  
Mitochondrial Potential ◽  
3C Protease ◽  
Wide Range

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

scholarly journals Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections.

1993 ◽  
Vol 41 (11) ◽  
pp. 1599-1604 ◽  
Author(s):  
S R Shi ◽  
B Chaiwun ◽  
L Young ◽  
R J Cote ◽  
C R Taylor
Keyword(s):  
Androgen Receptor ◽  
Enzymatic Digestion ◽  
Antigen Retrieval ◽  
Urea Solution ◽  
Paraffin Sections ◽  
Buffer Solution ◽  
Solution Ph ◽  
Citrate Buffer ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed

We developed a staining protocol for demonstration of androgen receptor (AR) in formalin-fixed, paraffin-embedded tissue sections. The method is based on the antigen retrieval microwave (MW) heating technique. Results are compared with different types of enzyme digestion pre-treatments. The strongest immunostaining signal and clearest background were obtained by MW heating of dewaxed paraffin sections in 5% urea or citrate buffer solution (pH 6); pure distilled water gave less consistent results. Enzymatic digestion with pepsin (0.05% in 2 N HCl) for 30 min at room temperature, or trypsin followed by pronase, or pronase digestion alone, also produced enhanced staining of AR in some cases, but there was more nonspecific background, and specific reactivity was less intense. The antigen retrieval MW method can be used to demonstrate AR epitope in prostate tissue after fixation in formalin for as long as 7 days. AR immunolocalization was also compared in frozen and paraffin sections processed from the same specimen of prostate carcinoma tissue and was found to be qualitatively and quantitatively similar. This study also provided new information concerning the basic principles of the antigen retrieval MW method that may be helpful in further development of this technique.

Pepsinogen C Expression in Tumors of Extragastric Origin

2000 ◽  
Vol 15 (2) ◽  
pp. 165-170 ◽  
Author(s):  
A.M. Merino ◽  
J. Vázquez ◽  
J.C. Rodríguez ◽  
R. Fernández ◽  
I. Quintela ◽  
...  
Keyword(s):  
Basal Cell ◽  
Immunohistochemical Staining ◽  
Proteolytic Enzyme ◽  
Human Tumors ◽  
Positive Staining ◽  
Human Tissues ◽  
Breast Carcinomas ◽  
Pepsinogen C ◽  
Human Carcinomas

We have examined by immunohistochemistry the ability of human carcinomas of various origin to produce pepsinogen C, an aspartyl proteinase mainly involved in the digestion of proteins in the stomach and recently found to be associated with breast carcinomas. Of the 268 tumors analyzed 80 (29.8%) showed positive staining for pepsinogen C. These positive tumors included 12 gastric (38.7% of the 31 examined cases), nine pancreatic (42.8%), two renal (20%), 12 prostatic (40%), three bladder (27.3%), 14 endometrial (29.7%) and 18 ovarian (40%) carcinomas. We also detected 10 melanomas (50%) that were positive for pepsinogen C. By contrast, immunohistochemical staining for the proteinase was not detected in colorectal, cervical, lung and basal cell skin carcinomas. These results demonstrate that pepsinogen C, a proteolytic enzyme of highly restricted expression in human tissues, can also be expressed by a wide variety of human carcinomas. In addition, and similar to pepsinogen C expression in breast carcinomas, the production of this enzyme by different human tumors might be related to putative hormonal alterations associated with the development and progression of these tumors.

STUDYING OF ACTIVITY OF INHIBITORS OF PROTEOLYTIC ENZYMES IN PRODUCTS OF PROCESSING OF GRAIN HARICOT (PHASEOLUS VULGARIS)

2018 ◽  
Author(s):  
Н.Т. ШАМКОВА ◽  
А.М. АБДУЛХАМИД
Keyword(s):  
Phaseolus Vulgaris ◽  
Liquid Medium ◽  
Proteolytic Enzymes ◽  
Trypsin Inhibitors ◽  
Milk Whey ◽  
Fine Grinding ◽  
Bean Flour ◽  
Wide Range ◽  
Mashed Potatoes

Определено содержание ингибиторов протеолитических ферментов в фасолевой муке, в пюре из зерновой фасоли, сваренной в воде, и в пюре из зерновой фасоли, сваренной после замачивания в воде в молочной сыворотке. Обосновано использование молочной сыворотки в качестве жидкой среды для варки зерновой фасоли после замачивания. Разработана технология полуфабриката в виде фасолевого пюре, предусматривающая замачивание фасоли в воде, варку в молочной сыворотке, грубое измельчение доведенной до готовности фасоли, последующее тонкое измельчение и охлаждение. Установлено, что в фасолевом пюре активность ингибиторов трипсина значительно ниже, чем в муке из фасоли, что делает пюре более предпочтительным полуфабрикатом для производства широкого ассортимента кулинарной продукции. The content of inhibitors of proteolytic enzymes in bean flour, in puree from beans harvested in water and in puree from cereal beans welded in milk whey after soaking in water is determined. The use of whey as a liquid medium for cooking grain beans after soaking is substantiated. The technology of semi-finished product in the form of bean puree, providing for soaking beans in water, cooking in milk whey, coarse grinding of the bean brought to the ready, subsequent fine grinding and cooling is developed. It has been found that the activity of trypsin inhibitors in bean puree is much lower than in bean flour, which makes mashed potatoes a more preferred semi-finished product for the production of a wide range of culinary products.

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