Robust Immunohistochemical Staining of Several Classes of Proteins in Tissues Subjected to Autolysis

Maleszewski, Joseph J.; Lu, Jie; Fox-Talbot, Karen; Halushka, Marc K.

doi:10.1369/jhc.6a7152.2007

Cited by 29 publications

(18 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We allowed the heart tissue to autolyze for 24 hours, either at ambient temperature or shifted to 4°C after 12 hours. We chose a limit of 24 hours as we have shown previously that protein expression remains intact for 24 hours by immunohistochemistry, but degradation is noticeable at 48 hours [36]. Thus our findings are applicable to a 24 hour time course and we cannot speculate on late harvesting.…”

Section: Resultsmentioning

confidence: 99%

Postmortem cardiac tissue maintains gene expression profile even after late harvesting

et al. 2012

View full text Add to dashboard Cite

BackgroundGene expression studies can be used to help identify disease-associated genes by comparing the levels of expressed transcripts between cases and controls, and to identify functional genetic variants (expression quantitative loci or eQTLs) by comparing expression levels between individuals with different genotypes. While many of these studies are performed in blood or lymphoblastoid cell lines due to tissue accessibility, the relevance of expression differences in tissues that are not the primary site of disease is unclear. Further, many eQTLs are tissue specific. Thus, there is a clear and compelling need to conduct gene expression studies in tissues that are specifically relevant to the disease of interest. One major technical concern about using autopsy-derived tissue is how representative it is of physiologic conditions, given the effect of postmortem interval on tissue degradation.ResultsIn this study, we monitored the gene expression of 13 tissue samples harvested from a rapid autopsy heart (non-failed heart) and 7 from a cardiac explant (failed heart) through 24 hours of autolysis. The 24 hour autopsy simulation was designed to reflect a typical autopsy scenario where a body may begin cooling to ambient temperature for ~12 hours, before transportation and storage in a refrigerated room in a morgue. In addition, we also simulated a scenario wherein the body was left at room temperature for up to 24 hours before being found. A small fraction (< 2.5%) of genes showed fluctuations in expression over the 24 hr period and largely belong to immune and signal response and energy metabolism-related processes. Global expression analysis suggests that RNA expression is reproducible over 24 hours of autolysis with 95% genes showing < 1.2 fold change. Comparing the rapid autopsy to the failed heart identified 480 differentially expressed genes, including several types of collagens, lumican (LUM), natriuretic peptide A (NPPA) and connective tissue growth factor (CTGF), which allows for the clear separation between failing and non-failing heart based on gene expression profiles.ConclusionsOur results demonstrate that RNA from autopsy-derived tissue, even up to 24 hours of autolysis, can be used to identify biologically relevant expression pattern differences, thus serving as a practical source for gene expression experiments.

show abstract

Section: Resultsmentioning

confidence: 99%

Postmortem cardiac tissue maintains gene expression profile even after late harvesting

et al. 2012

View full text Add to dashboard Cite

show abstract

“…In forensic casework, however, postmortem changes are often unpredictable; thus, the quality of tissue specimens should be evaluated at least on routine histology. Further investigation is needed for the stability of these biomarkers in cases of longer postmortem interval; in such cases, Western blotting may be useful for precise evaluation of the chemical and immunohistochemical integrity of target biomarkers [42].…”

Section: Discussionmentioning

confidence: 99%

Quantitative immunohistochemical analysis of human brain basic fibroblast growth factor, glial fibrillary acidic protein and single-stranded DNA expressions following traumatic brain injury

Wang

Ishikawa

Michiue

et al. 2012

Forensic Science International

View full text Add to dashboard Cite

“…Immunohistochemistry and Routine Staining IHC was performed using standard protocols described previously (Maleszewski et al 2007). Briefly, slides were cut from each TMA block and kept in an airtight container at 220C until use.…”

Section: Tissue Microarray Creationmentioning

confidence: 99%

Glomerular Protein Levels of Matrix Metalloproteinase-1 and Tissue Inhibitor of Metalloproteinase-1 Are Lower in Diabetic Subjects

Cornish

Bagnasco

Macgregor

et al. 2009

J Histochem Cytochem.

Self Cite

View full text Add to dashboard Cite

S U M M A R Y Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix turnover throughout the body, including in renal glomeruli. We investigated protein levels of multiple MMPs (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 in glomeruli and investigated whether disease phenotypes were associated with levels of these proteins. Renal cortex was collected from 100 adult autopsy subjects arrayed across 17 tissue microarrays. Immunohistochemical staining intensity for each MMP and TIMP-1 was determined using quantitative color deconvolution techniques. We observed significantly decreased glomerular MMP-1 and TIMP-1 staining in subjects with diabetes, hypertension, and an estimated glomerular filtration rate ,30 ml/min/1.73 m 2 in univariate analyses. MMP-1 staining, but not TIMP-1 staining, was inversely correlated with increased glomerular fibrosis (r 5 20.40). In multivariable analysis, diabetes was robustly associated with decreased staining intensity. This study indicates that in human subjects, the long-term sequelae of diseases such as diabetes that cause chronic renal failure result in decreased TIMP-1 and MMP-1 proteins in renal glomeruli. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. . When an imbalance between activities of these two protein classes occurs, the result is often a pathological alteration of ECM. Numerous MMPs and TIMPs, including MMP-1, -2, -3, -9, and -13 and TIMP-1, -2, and -3 have been investigated within the kidney and implicated in progressive renal scarring (Lenz et al. 2000;Ahmed et al. 2007). Altered renal MMP and TIMP activity results in increased ECM deposition and/or decreased ECM breakdown (Lenz et al. 2000). Within the glomeruli, MMPs have been implicated in pathological matrix accumulation. Both non-inflammatory and inflammatory glomerular diseases alter MMP and TIMP expression levels, often in opposite directions (Reckelhoff et al. 1993;Nakamura et al. 1994Nakamura et al. ,1995Singhal et al. 1996). Despite numerous animal models that describe MMP and TIMP alterations in glomeruli, there have been very few studies in human subjects. We undertook this study to examine glomerular MMP and TIMP protein levels in kidneys taken from 100 adults with a broad range of chronic diseases. We hypothesized that hypertension and diabetes would be associated with lower MMP and higher TIMP protein levels, based on animal data. We used vascular tissue microarrays (TMAs) and color deconvolution techniques to characterize the amount of each protein in the glomeruli and correlated these measures with clinical variables (Cornish and Halushka in press; Halushka et al. in press).

show abstract

Robust Immunohistochemical Staining of Several Classes of Proteins in Tissues Subjected to Autolysis

Cited by 29 publications

References 31 publications

Postmortem cardiac tissue maintains gene expression profile even after late harvesting

Postmortem cardiac tissue maintains gene expression profile even after late harvesting

Quantitative immunohistochemical analysis of human brain basic fibroblast growth factor, glial fibrillary acidic protein and single-stranded DNA expressions following traumatic brain injury

Glomerular Protein Levels of Matrix Metalloproteinase-1 and Tissue Inhibitor of Metalloproteinase-1 Are Lower in Diabetic Subjects

Contact Info

Product

Resources

About