Role of microwave heating in antigen retrieval in cryosections of the formalin-fixed mammalian tissues

Background: Breast cancer (BC) is a well-documented major cause of female morbidity and mortality worldwide. Ongoing research era is focusing on the establishment of diagnostic and prognostic markers, helping for early pick up of the cases, proper prognosis evaluation and clarifying reliable treatment strategy. Aim of the Study: This study aimed to evaluate the role of Ki-67 as prognostic marker for breast cancer in Egyptian females population. Patients and Methods: 120 BC patients and 30 age and BMI matching health controls are the subjects of the study, Ki-67 index values were investigated by immunohistochemistry that was performed on 5-lm slides of formalin-fixed and paraffin-embedded archival tumor tissue (core needle biopsy samples). Antigen retrieval was performed in a micro-oven in citrate buffer pH 6 for 20 minutes. Ki-67–stained slides were captured digitally at a hot spot at 3200 magnification. The Ki-67 labeling index was measured using digital image analysis software. Image analysis was performed by an experienced pathologist. Ki-67 index values were correlated with the clinicopathologic aspects of the BC patients. Results: Our study showed that Ki-67 index values revealed gradual increase with disease severity and correlated with poor prognosis aspects. Conclusion: Ki-67 index values are shown to be associated with breast cancer prognosis, supporting their role as prognostic biomarkers.

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A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed, paraffin embedded tissues

Biotechnic & Histochemistry ◽

10.1080/10520290310001593775 ◽

2003 ◽

Vol 78 (2) ◽

pp. 119-128 ◽

Cited By ~ 20

Author(s):

Z Kahveci ◽

Fz Minbay ◽

S Noyan ◽

I Çavusoglu

Keyword(s):

Microwave Heating ◽

Antigen Retrieval ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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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.

Journal of Histochemistry & Cytochemistry ◽

10.1177/43.4.7534785 ◽

1995 ◽

Vol 43 (4) ◽

pp. 429-437 ◽

Cited By ~ 53

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.

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Comprehensive Evaluation of PAXgene Fixation on Oral Cancer Tissues Using Routine Histology, Immunohistochemistry, and FTIR Microspectroscopy

Biomolecules ◽

10.3390/biom11060889 ◽

2021 ◽

Vol 11 (6) ◽

pp. 889

Author(s):

Pooja Lahiri ◽

Suranjana Mukherjee ◽

Biswajoy Ghosh ◽

Debnath Das ◽

Basudev Lahiri ◽

...

Keyword(s):

Comprehensive Evaluation ◽

Antigen Retrieval ◽

Tissue Fixation ◽

Ftir Microspectroscopy ◽

Protein Secondary Structures ◽

Associated Proteins ◽

Cancer Tissues ◽

Formalin Fixed ◽

Fixation System

The choice of tissue fixation is critical for preserving the morphology and biochemical information of tissues. Fragile oral tissues with lower tensile strength are challenging to process for histological applications as they are prone to processing damage, such as tissue tear, wrinkling, and tissue fall-off from slides. This leads to loss of morphological information and unnecessary delay in experimentation. In this study, we have characterized the new PAXgene tissue fixation system on oral buccal mucosal tissue of cancerous and normal pathology for routine histological and immunohistochemical applications. We aimed to minimize the processing damage of tissues and improve the quality of histological experiments. We also examined the preservation of biomolecules by PAXgene fixation using FTIR microspectroscopy. Our results demonstrate that the PAXgene-fixed tissues showed significantly less tissue fall-off from slides. Hematoxylin and Eosin staining showed comparable morphology between formalin-fixed and PAXgene-fixed tissues. Good quality and slightly superior immunostaining for cancer-associated proteins p53 and CK5/6 were observed in PAXgene-fixed tissues without antigen retrieval than formalin-fixed tissues. Further, FTIR measurements revealed superior preservation of glycogen, fatty acids, and amide III protein secondary structures in PAXgene-fixed tissues. Overall, we present the first comprehensive evaluation of the PAXgene tissue fixation system in oral tissues. This study concludes that the PAXgene tissue fixation system can be applied to oral tissues to perform diagnostic molecular pathology experiments without compromising the quality of the morphology or biochemistry of biomolecules.

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

Journal of Histochemistry & Cytochemistry ◽

10.1177/41.11.7691930 ◽

1993 ◽

Vol 41 (11) ◽

pp. 1599-1604 ◽

Cited By ~ 197

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.

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Role of pre-ionization in NBI plasma start-up of Heliotron J using non-resonant microwave heating

Nuclear Fusion ◽

10.1088/1741-4326/ac2105 ◽

2021 ◽

Vol 61 (11) ◽

pp. 116009

Author(s):

S. Kobayashi ◽

K. Nagasaki ◽

K. Hada ◽

T. Stange ◽

H. Okada ◽

...

Keyword(s):

Microwave Heating ◽

Start Up

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The role of mitochondrial Ca2+ transport and matrix Ca2+ in signal transduction in mammalian tissues

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/0005-2728(90)90269-a ◽

1990 ◽

Vol 1018 (2-3) ◽

pp. 287-291 ◽

Cited By ~ 67

Author(s):

James G. McCormack ◽

Richard M. Denton

Keyword(s):

Signal Transduction ◽

Mammalian Tissues

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Prognostic value of dickkopf-1 and ß-catenin expression according to the antitumor immunity of CD8-positive tumor-infiltrating lymphocytes in biliary tract cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e16172 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e16172-e16172

Author(s):

Seoree Kim ◽

Yoon Ho Ko ◽

Hye Sung Won ◽

Ji Hyun Yang ◽

Der Sheng Sun ◽

...

Keyword(s):

Clinical Outcomes ◽

Biliary Tract ◽

Biliary Tract Cancer ◽

Cox Regression ◽

Research Network ◽

Antigen Retrieval ◽

Tract Cancer ◽

Tissue Sections ◽

Dkk1 Expression

e16172 Background: Despite recent advancements in the understanding of the molecular biology of biliary tract cancer (BTC), target therapy and immunotherapy have demonstrated only limited efficacy, with cytotoxic systemic therapy still being the most effective treatment in BTC, except for surgery. Thus, this study aimed to analyze the role of DKK1 or β-catenin as a prognostic factor in BTC and determine the clinical association of ß-catenin and DKK1 with CD8+ tumor-infiltrating lymphocyte (TIL). Methods: We used data in The Cancer Genome Atlas (TCGA) Research Network and the clinicopathological data of 145 patients with BTC who had undergone primary radical resection between 2006 and 2016. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections, and whole tissue sections of representative tumor samples were used for antigen retrieval. Results: CD8+TIL expression was a significant predictor of favorable overall survival (OS) and recurrence-free survival (RFS) (median OS, 34.9months in TIL-high, 16.7months in TIL-low, P < 0.0001 respectively; median RFS, 27.1months in TIL-high, 10months in TIL-low, P < 0.0001 respectively). Positive ß-catenin expression and high DKK1 expression was also associated with a shorter OS (median OS, 23.95months in positive ß-catenin, 26.1months in negative ß-catenin, P = 0.1009 respectively; median OS, 19.4months in high DKK1, 31.65months in Low DKK1, P = 0.0093 respectively), but not RFS (p = 0.1466, at ß-catenin respectively; p = 0.2924, in DKK1 respectively). In the CD8+TIL-high BTC group, the tumor expression of β-catenin and DKK1 had a significant negative impact on either OS or RFS (p = 0.0146 and p = 0.0112, at ß-catenin respectively; p = 0.0950 and p = 0.3904, in DKK1 respectively). However, in the TIL-low BTC group, there were no differences in OS or RFS according to ß-catenin and DKK1 expression (p = 0.5108 and p = 0.8431, at ß-catenin respectively; p = 0.1127 and p = 0.1095, in DKK1 respectively). Cox regression multivariate analysis demonstrated that CD8+ TIL (hazard ratio [HR], 0.490; 95% confidence interval (CI), 0.303-0.791; p = 0.004) and β-catenin (HR, 1.652; 95% CI, 1.035-2.639; p = 0.036) retained significant association with OS after adjustment for all variables. Conclusions: Among patients with resected BTC, β-catenin and DKK1 protein levels are associated with poor clinical outcomes, whereas high CD8+ TIL levels are associated with good clinical outcomes. This confirms the differential clinical role of Wnt/β-catenin and DKK1 proteins according to TIL expression in BTC.

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Loss of keratin 6 (K6) proteins reveals a function for intermediate filaments during wound repair

The Journal of Cell Biology ◽

10.1083/jcb.200305032 ◽

2003 ◽

Vol 163 (2) ◽

pp. 327-337 ◽

Cited By ~ 146

Author(s):

Pauline Wong ◽

Pierre A. Coulombe

Keyword(s):

Gene Expression ◽

Wound Repair ◽

Tissue Injury ◽

Blood Clot ◽

Explant Culture ◽

Null Mouse ◽

Actin Organization ◽

Mammalian Tissues

The ability to heal wounds is vital to all organisms. In mammalian tissues, alterations in intermediate filament (IF) gene expression represent an early reaction of cells surviving injury. We investigated the role of keratin IFs during the epithelialization of skin wounds using a keratin 6α and 6β (K6α/K6β)-null mouse model. In skin explant culture, null keratinocytes exhibit an enhanced epithelialization potential due to increased migration. The extent of the phenotype is strain dependent, and is accompanied by alterations in keratin IF and F-actin organization. However, in wounded skin in vivo, null keratinocytes rupture as they attempt to migrate under the blood clot. Fragility of the K6α/K6β-null epidermis is confirmed when applying trauma to chemically treated skin. We propose that the alterations in IF gene expression after tissue injury foster a compromise between the need to display the cellular pliability necessary for timely migration and the requirement for resilience sufficient to withstand the rigors of a wound site.

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