scholarly journals ­Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue

2016 ◽  
Author(s):  
Gretchen H Delcambre ◽  
Junjie Liu ◽  
Jenna M Herrington ◽  
Kelsey Vallario ◽  
Maureen T Long
Keyword(s):  
Polyclonal Antibodies ◽  
Inflammatory Cells ◽  
Specific Protein ◽  
Proteinase K ◽  
Antigen Retrieval ◽  
Phenotypic Characterization ◽  
Buffer Solution ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Species Specific

Phenotypic characterization of cellular responses in equine infectious encephalitides has had limited description of both peripheral and resident cell populations in central nervous system (CNS) tissues due to limited species-specific reagents that react with formalin-fixed, paraffin embedded tissue (FFPE). This study identified a set of antibodies for investigating the immunopathology of infectious CNS diseases in horses. Multiple commercially available staining reagents and antibodies derived from antigens of various species for manual immunohistochemistry (IHC) were screened. Several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization-detection systems were tested during IHC protocol development. Boiling of slides in a low pH, citrate-based buffer solution in a double-boiler system was most consistent for epitope retrieval. Pressure-cooking, microwaving, high pH buffers, and proteinase K solutions often resulted in tissue disruption or no reactivity. Optimal blocking reagents and concentrations of each working antibody were determined. Ultimately, a set of monoclonal (mAb) and polyclonal antibodies (pAb) were identified for CD3+ (pAb A0452, Dako) T-lymphocytes, CD79αcy+ B-lymphocytes (mAb HM57, Dako), macrophages (mAb MAC387, Leica), NF-H+ neurons (mAb NAP4, EnCor Biotechnology), microglia/macrophage (pAb Iba-1, Wako), and GFAP+ astrocytes (mAb 5C10, EnCor Biotechnology). In paraffin embedded tissues, mAbs and pAbs derived from human and swine antigens were very successful at binding equine tissue targets. Individual, optimized protocols are provided for each positively reactive antibody for analyzing equine neuroinflammatory disease histopathology.

scholarly journals Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1601 ◽  
Author(s):  
Gretchen H. Delcambre ◽  
Junjie Liu ◽  
Jenna M. Herrington ◽  
Kelsey Vallario ◽  
Maureen T. Long
Keyword(s):  
Polyclonal Antibodies ◽  
Inflammatory Cells ◽  
Specific Protein ◽  
Proteinase K ◽  
Antigen Retrieval ◽  
Phenotypic Characterization ◽  
Buffer Solution ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Species Specific

Phenotypic characterization of cellular responses in equine infectious encephalitides has had limited description of both peripheral and resident cell populations in central nervous system (CNS) tissues due to limited species-specific reagents that react with formalin-fixed, paraffin embedded tissue (FFPE). This study identified a set of antibodies for investigating the immunopathology of infectious CNS diseases in horses. Multiple commercially available staining reagents and antibodies derived from antigens of various species for manual immunohistochemistry (IHC) were screened. Several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization-detection systems were tested during IHC protocol development. Boiling of slides in a low pH, citrate-based buffer solution in a double-boiler system was most consistent for epitope retrieval. Pressure-cooking, microwaving, high pH buffers, and proteinase K solutions often resulted in tissue disruption or no reactivity. Optimal blocking reagents and concentrations of each working antibody were determined. Ultimately, a set of monoclonal (mAb) and polyclonal antibodies (pAb) were identified for CD3+(pAb A0452, Dako) T-lymphocytes, CD79αcy+B-lymphocytes (mAb HM57, Dako), macrophages (mAb MAC387, Leica), NF-H+neurons (mAb NAP4, EnCor Biotechnology), microglia/macrophage (pAb Iba-1, Wako), and GFAP+astrocytes (mAb 5C10, EnCor Biotechnology). In paraffin embedded tissues, mAbs and pAbs derived from human and swine antigens were very successful at binding equine tissue targets. Individual, optimized protocols are provided for each positively reactive antibody for analyzing equine neuroinflammatory disease histopathology.

scholarly journals ­Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue

2016 ◽  
Author(s):  
Gretchen H Delcambre ◽  
Junjie Liu ◽  
Jenna M Herrington ◽  
Kelsey Vallario ◽  
Maureen T Long
Keyword(s):  
Polyclonal Antibodies ◽  
Inflammatory Cells ◽  
Specific Protein ◽  
Proteinase K ◽  
Antigen Retrieval ◽  
Phenotypic Characterization ◽  
Buffer Solution ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Species Specific

Phenotypic characterization of cellular responses in equine infectious encephalitides has had limited description of both peripheral and resident cell populations in central nervous system (CNS) tissues due to limited species-specific reagents that react with formalin-fixed, paraffin embedded tissue (FFPE). This study identified a set of antibodies for investigating the immunopathology of infectious CNS diseases in horses. Multiple commercially available staining reagents and antibodies derived from antigens of various species for manual immunohistochemistry (IHC) were screened. Several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization-detection systems were tested during IHC protocol development. Boiling of slides in a low pH, citrate-based buffer solution in a double-boiler system was most consistent for epitope retrieval. Pressure-cooking, microwaving, high pH buffers, and proteinase K solutions often resulted in tissue disruption or no reactivity. Optimal blocking reagents and concentrations of each working antibody were determined. Ultimately, a set of monoclonal (mAb) and polyclonal antibodies (pAb) were identified for CD3+ (pAb A0452, Dako) T-lymphocytes, CD79αcy+ B-lymphocytes (mAb HM57, Dako), macrophages (mAb MAC387, Leica), NF-H+ neurons (mAb NAP4, EnCor Biotechnology), microglia/macrophage (pAb Iba-1, Wako), and GFAP+ astrocytes (mAb 5C10, EnCor Biotechnology). In paraffin embedded tissues, mAbs and pAbs derived from human and swine antigens were very successful at binding equine tissue targets. Individual, optimized protocols are provided for each positively reactive antibody for analyzing equine neuroinflammatory disease histopathology.

scholarly journals NeuN: a useful neuronal marker for diagnostic histopathology.

1996 ◽  
Vol 44 (10) ◽  
pp. 1167-1171 ◽  
Author(s):  
H K Wolf ◽  
R Buslei ◽  
R Schmidt-Kastner ◽  
P K Schmidt-Kastner ◽  
T Pietsch ◽  
...  
Keyword(s):  
Neuronal Cell ◽  
Cell Types ◽  
Specific Protein ◽  
Antigen Retrieval ◽  
Neuronal Marker ◽  
Formalin Fixed Paraffin ◽  
Wide Range ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed ◽  
Diagnostic Histopathology

The monoclonal antibody A60 specifically recognizes the DNA-binding, neuron-specific protein NeuN, which is present in most neuronal cell types of vertebrates. In this study we demonstrate the potential use of NeuN as a diagnostic neuronal marker using a wide range of formalin-fixed, paraffin-embedded human surgical and autopsy specimens from the central and peripheral nervous system. After microwave antigen retrieval, almost all neuronal populations revealed strong immunoreactivity for NeuN in nuclei, perikarya, and some proximal neuronal processes, whereas more distal axon cylinders and dendritic ramifications were not stained. The stain greatly enhanced the gray matter architecture. NeuN immunoreactivity was not detected in Purkinje cells, most neurons of the internal nuclear layer of the retina, and in sympathetic chain ganglia. We examined nine gangliogliomas and 14 dysembryoplastic neuroepithelial tumors, one ganglioneuroma, and one dysplastic cerebellar gangliocytoma. The neuronal component of all of these lesions showed marked immunoreactivity for NeuN. In addition, NeuN immunoreactivity was focally seen in one of seven medulloblastomas with prominent neuronal differentiation. There was no staining of non-neuronal structures. The results indicate that NeuN immunoreactivity is a sensitive and specific neuronal marker in formalin-fixed, paraffin-embedded tissues, and may be useful in diagnostic histopathology.

scholarly journals Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

1991 ◽  
Vol 39 (6) ◽  
pp. 741-748 ◽  
Author(s):  
S R Shi ◽  
M E Key ◽  
K L Kalra
Keyword(s):  
Polyclonal Antibodies ◽  
Antigen Retrieval ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Pre Treatment ◽  
Immunohistochemical Techniques ◽  
Formalin Fixed

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.

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.

scholarly journals Maximizing RNA Yield from Archival Renal Tumors and Optimizing Gene Expression Analysis

2009 ◽  
Vol 15 (1) ◽  
pp. 80-85 ◽  
Author(s):  
Sean T. Glenn ◽  
Karen L. Head ◽  
Bin T. Teh ◽  
Kenneth W. Gross ◽  
Hyung L. Kim
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reverse Transcription ◽  
Gene Expression Analysis ◽  
Rna Isolation ◽  
Proteinase K ◽  
Specific Primers ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan ® PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure™ kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen’s TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers’ protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan ® PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan® qPCR can be optimized by using the MasterPure™ RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription.

scholarly journals Monoclonal and Polyclonal Antibodies for Immunohistochemical Detection of Bovine Parainfluenza Type 3 Virus in Frozen and Formalin-Fixed Paraffin-Embedded Tissues

1992 ◽  
Vol 4 (4) ◽  
pp. 393-399 ◽  
Author(s):  
Deborah M. Haines ◽  
Jane C. Kendall ◽  
Brad W. Remenda ◽  
Michelle M. Breker-Klassen ◽  
Edward G. Clark
Keyword(s):  
Polyclonal Antibodies ◽  
Accurate Identification ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Type 3 ◽  
Murine Monoclonal Antibodies ◽  
Formalin Fixed ◽  
Immunohistochemical Testing

Accurate identification of bovine Parainfluenza type 3 virus in bovine respiratory disease requires dependable, sensitive, and specific techniques for detection in affected animals. Immunohistochemical testing can be a rapid and reliable means of demonstration of virus in tissues from suspect cases; however, this procedure is dependent upon the quality of the antisera directed against the viral antigens. The production of rabbit polyclonal and murine monoclonal antibodies directed against bovine Parainfluenza type 3 virus and techniques for their use in fresh-frozen and formalin-fixed paraffin-embedded tissues in immunofluorescence and immunoperoxidase-based immunohistochemical tests are described.

scholarly journals Pathology Quality Control for Multiplex Immunofluorescence and Image Analysis Assessment in Longitudinal Studies

2021 ◽  
Vol 8 ◽  
Author(s):  
Rossana Lazcano ◽  
Frank Rojas ◽  
Caddie Laberiano ◽  
Sharia Hernandez ◽  
Edwin Roger Parra
Keyword(s):  
Quality Control ◽  
Image Analysis ◽  
Inflammatory Cells ◽  
Analysis Data ◽  
Malignant Cells ◽  
Core Needle ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Organ Site ◽  
Immune Profiling

Immune profiling of formalin-fixed, paraffin-embedded tissues using multiplex immunofluorescence (mIF) staining and image analysis methodology allows for the study of several biomarkers on a single slide. The pathology quality control (PQC) for tumor tissue immune profiling using digital image analysis of core needle biopsies is an important step in any laboratory to avoid wasting time and materials. Although there are currently no established inclusion and exclusion criteria for samples used in this type of assay, a PQC is necessary to achieve accurate and reproducible data. We retrospectively reviewed PQC data from hematoxylin and eosin (H&E) slides and from mIF image analysis samples obtained during 2019. We reviewed a total of 931 reports from core needle biopsy samples; 123 (13.21%) were excluded during the mIF PQC. The most common causes of exclusion were the absence of malignant cells or fewer than 100 malignant cells in the entire section (n = 42, 34.15%), tissue size smaller than 4 × 1 mm (n = 16, 13.01%), fibrotic tissue without inflammatory cells (n = 12, 9.76%), and necrotic tissue (n = 11, 8.94%). Baseline excluded samples had more fibrosis (90 vs 10%) and less necrosis (5 vs 90%) compared with post-treatment excluded samples. The most common excluded organ site of the biopsy was the liver (n = 19, 15.45%), followed by soft tissue (n = 17, 13.82%) and the abdominal region (n = 15, 12.20%). We showed that the PQC is an important step for image analysis and that the absence of malignant cells is the most limiting sample characteristic for mIF image analysis. We also discuss other challenges that pathologists need to consider to report reliable and reproducible image analysis data.

PD-L1 Expression on Cell Block Preparations of Pancreatic Adenocarcinoma

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S94-S95
Author(s):  
Victoria Costa ◽  
David Kim ◽  
Melanie Johncilla ◽  
Abha Goyal ◽  
Rema Rao
Keyword(s):  
Disease Progression ◽  
Tumor Cells ◽  
Locally Advanced ◽  
Immunohistochemical Analysis ◽  
Inflammatory Cells ◽  
Fixation Method ◽  
Lung Cancers ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Combined Positive Score

Abstract Objectives Programmed death-ligand 1 (PD-L1) is an emerging molecular target in anticancer therapy, most notably non–small cell lung cancers. PD-L1 expression in pancreatic adenocarcinomas (PDAs) on surgical specimens is highly variable, with 10% to 60% of tumors showing expression. PD-L1 expression in PDA on endoscopic ultrasound-guided fine needle aspirations (EUS-FNAs) has been rarely studied. Methods Formalin-fixed, paraffin embedded (FFPE) cell blocks (CBs) from 65 EUS-FNA samples of 55 patients, with a diagnosis of PDA, with at least 20% tumor cellularity were retrieved. The cell blocks were originally fixed in CytoRich fixative. Immunohistochemical staining (IHC) for PD-L1 was performed using the M365329-1 (22C3) clone according to the manufacturer’s approved protocol and optimized for the fixation method using appropriate controls. A combined positive score (CPS) defined per CAP guidelines as the total number of positive tumor cells and inflammatory cells as a percentage of the total number of tumor cells was assessed for each case and was grouped as <1, 1-20, or >20. Results Twenty-five samples (38%) from 21 patients showed immunoreactivity to PD-L1, with 21 (32%) having a CPS of <1 and four (6%) having a CPS of 1-20. Eight of these 25 samples had surgical correlates, of which concordant staining was noted in five (62.5%). Of the discordant three, decreased tumor cell sampling in the core biopsy was noted in one. Overall, 20 of 21 patients (95%) with PD-L1 immunoreactivity had disease progression with 17 (81%) associated with metastatic disease and three (14%) with locally advanced disease. Conclusion Immunohistochemical analysis for PD-L1 is feasible on EUS-FNA CB samples when optimized and validated for the fixation method using appropriate controls. PD-L1 expression is seen in only a minority of PDAs, albeit with a very low CPS. The potential role of PD-L1 as a prognostic marker for disease progression needs further exploration.

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