Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using ''filter aided sample preparation'' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on ''pipette-tip'' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.     

Immunofluorescent Staining of Epon Embedded Kidney Sections Through Simultaneous use of Two Different Fluorochrome-Conjugated Antisera

Kidney biopsies can be examined in Epon sections for comparison of immunofluorescence and histology. This is possible by an incubation method which has now been modified to allow simultaneous localization of two antigens using fluorescein and rhodamine-conjugated antibodies on the same semithin sections of formalin fixed tissue. Consecutive sections from the same blocks can also be cut for electron microscopy. The method is now used in our immunopathological diagnostic procedures for examination of kidney biopsies.     

High-Throughput Sequencing and Copy Number Variation Detection Using Formalin Fixed Embedded Tissue in Metastatic Gastric Cancer

In the era of targeted therapy, mutation profiling of cancer is a crucial aspect of making therapeutic decisions. To characterize cancer at a molecular level, the use of formalin-fixed paraffin-embedded tissue is important. We tested the Ion AmpliSeq Cancer Hotspot Panel v2 and nCounter Copy Number Variation Assay in 89 formalin-fixed paraffin-embedded gastric cancer samples to determine whether they are applicable in archival clinical samples for personalized targeted therapies. We validated the results with Sanger sequencing, real-time quantitative PCR, fluorescence in situ hybridization and immunohistochemistry. Frequently detected somatic mutations included TP53 (28.17%), APC (10.1%), PIK3CA (5.6%), KRAS (4.5%), SMO (3.4%), STK11 (3.4%), CDKN2A (3.4%) and SMAD4 (3.4%). Amplifications of HER2, CCNE1, MYC, KRAS and EGFR genes were observed in 8 (8.9%), 4 (4.5%), 2 (2.2%), 1 (1.1%) and 1 (1.1%) cases, respectively. In the cases with amplification, fluorescence in situ hybridization for HER2 verified gene amplification and immunohistochemistry for HER2, EGFR and CCNE1 verified the overexpression of proteins in tumor cells. In conclusion, we successfully performed semiconductor-based sequencing and nCounter copy number variation analyses in formalin-fixed paraffin-embedded gastric cancer samples. High-throughput screening in archival clinical samples enables faster, more accurate and cost-effective detection of hotspot mutations or amplification in genes.     

Immunoperoxidase Staining for Estrogen and Progesterone Receptors in Archival Formalin Fixed, Paraffin Embedded Breast Carcinomas after Microwave Antigen Retrieval

Immunoperoxidase staining was performed for estrogen and progesterone receptors in 93 cases of primary breast carcinoma. Breast tumor samples were fixed in formalin and embedded in paraffin. Antigen retrieval was performed by microwave heating in citrate buffer, pH 6.0, using precisely defined and reproducible conditions. The cases studied included material from the current year and from paraffin blocks retrieved from archival storage dating back to 1981. In all cases, estrogen and progesterone receptor values determined by biochemical assay were available for comparison with the immunohistochemical results. We found 94% agreement of results between the two methods.     

Reliable LC3 and p62 Autophagy Marker Detection in Formalin Fixed Paraffin Embedded Human Tissue by Immunohistochemistry

Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and nonneoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60-0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.Key words: Immunohistochemistry, immunofluorescence, formalin fixed paraffin embedded tissue, LC3B, p62, cancer     

Staining Nerve Fibers with Silver in Tissue Fixed with Bouin's Fluid

Nerve fibers, in organs fixed with Bouin's fluid, are usually refractive to the Davenport silver technic. The axons, however, can be successfully stained if the sections, on slides, are given a preliminary treatment with concentrated pyridine (1 hour), then a 24-hour bath of ammoniated alcohol (99 cc. 80% alcohol, 1 cc 28% ammonium hydroxide) and an interval in 40% aqueous silver nitrate (6-8 hours) before being immersed in the acidified alcoholic silver solution of Davenport. Following the silvering, reduction and toning of the axons, according to the procedure of Davenport, the surrounding non-nervous tissue elements can be counterstained with a combination of either azocarmine, light green and orange G, or azocarmine, aniline blue and orange G.     

Stabilization of Fluorochromes After Immunofluorescent Staining of Tissue and Embedding in Epon

Tissue blocks or sections immunofluo-nt stnined before embedding, ig., liver and kidney, can be stored for more than 3 years without demonstrable fluorescence decay. The processing steps, including poststaining dehydration by alcohols and embedding in expoxy resins, seem to stabilize the fluorochromes fluo-in isotbiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) so that they fade I- during illumination. This is an advantage of the preembedding, immunofluo-nt staining technique which is combined with a lack of damage to the antigens by the plastic embedding medium.     

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section¹. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).     

Staining of Different Endocrine Cells with Hydrochloric Acid-Toluidine Blue in Epon Embedded Rat Tissue

The usual HCl-toluidine blue staining of different endocrine cells is applicable to paraffin embedded material. A modification for Epon embedded tissue suitable for consecutive light and electron microscopic studies is described which makes it possible to find the same stained cell, both in a semithin section and in subsequent ultrathin sections. This method facilitates the search for scattered specific endocrine cells. Without removing the resin, sections of Epon embedded tissues were hydrolyzed for 17 hr in 1% HCl at 65 C and stained for 2 turn 0.1% toluidine blue in McIlvaine buffer, pH 5.8. The following cells were stained: C cells in thyroid glands; A and D cells in pancreatic islets; B cells in anterior pituitary; G, D and Ec cells in the gastrointestinal tract; Ad cells of the adrenal medulla.     

福尔马林对固定标本DNA提取和扩增的影响

福尔马林被广泛应用于生物标本的长期保存。由于福尔马林可能影响标本DNA的质量,因此需要对福尔马林固定标本DNA的提取和扩增过程进行改进。影响从福尔马林保存标本中提取的DNA质量的主要因素包括福尔马林导致的DNA与蛋白质之间、蛋白质与蛋白质之间、DNA与DNA之间的交联,福尔马林溶液的化学成分、pH值及浓度,标本保存的时间和温度,标本保存部位等。本文总结了目前常用的对标本DNA提取和扩增过程的改进措施及其优点。     

Novel Fixation of Plant Tissue, Staining through Paraffin with Alcian Blue and Hematoxylin, and Improved Slide Preparation

Onion (Allium cepa) root tips were fixed in a proprietary solution without aldehyde, toxic metals or acetic acid. Fixed specimens were embedded in paraffin, sectioned on a rotary microtome and mounted on detergent-washed slides without adhesive. Slides with ribbon segments affixed were immersed in 0.2% aqueous alcian blue 8GX in screw-capped Coplin jars in a water bath at 50 C for 1 hr. Excess alcian blue was rinsed off under cold running tap water and the slides were immersed in quick-mixed hematoxylin at room temperature for 15 min. Stained slides were deparaffinized, rinsed with isopropanol, air dried, and coverslips were affixed with resin. Thus, the traditional paraffin microtechnique has been modified at all steps from fixation to finishing slides with coverslips.     

Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity

In situ zymography is a method for the detection and localization of enzymatic activity in tissue sections. This method is used with frozen sections because routine fixation of tissue in neutral-buffered formalin inhibits enzyme activity. However, frozen sections present with poor tissue morphology, making precise localization of enzymatic activity difficult to determine. Ethanol- and zinc-buffered fixative (ZBF) are known to preserve both morphological and functional properties of the tissue well, but it has not previously been shown that these fixatives preserve enzyme activity. In the present study, we show that in situ zymography can be performed on ethanol- and ZBF-fixed paraffin-embedded tissue. Compared with snap-frozen tissue, ethanol- and ZBF-fixed tissue showed stronger signals and superior morphology, allowing for a much more precise detection of gelatinolytic activity. Gelatinolytic enzymes could also be extracted from both ethanol- and ZBF-fixed tissue. The yield, as analyzed by SDS-PAGE gelatin zymography and Western blotting, was influenced by the composition of the extraction buffer, but was generally lower than that obtained from unfixed tissue. (J Histochem Cytochem 58:29–39, 2010)     

Selective Staining by the Fluorochrome 5,5'-Diphenyl-9-ethyl-oxacarbocyanine. II. Application to Paraffin Embedded Nervous Tissue

We present a simple and rapid method to label specific structures of neural tissue in paraffin sections. After incubation of deparaffinized sections in the cyanine dye 5,5'-diphenyl-9-ethyl-oxacarbocyanine (DEOC), three different washing and mounting procedures were performed. Incubation in DEOC followed by washes in ethanol and water and mounting in glycerol-gelatin resulted in selective labeling of myelin in the central and peripheral nervous systems. Weak labeling of myelin and axons and staining of nuclei of neurons was seen after incubation in DEOC followed by washes in ethanol and xylene, and mounting in Eukitt. Nuclear proteins, the endoplasmic reticulum and the Golgi apparatus were stained strongly and exclusively after incubation in DEOC, washes in water, ethanol and xylene, and mounting in Eukitt. Thus the absorption pattern of DEOC is changed significantly by solvents applied after the fluorochrome. In any case, fluorescence did not fade even after repeated and intense fluorescence microscopy over a period of 6 months.     

Staining Nervous Tissue with Phloxine and Gallocyanin in Sequence

In the examination of monkey brain and spinal cord for neurovirulence in connection with the production of live poliovirus vaccine, sections must show clearly the details of the nerve cell bodies, and at the same time the identity of any inflammatory elements. The intact neuronal elements stain well with gallocyanin. Degenerated nerve cells and inflammatory cells stain well with eosin-type stains.     

Staining of Oligodendroglia with Silver Ammino Tungstate in Unembedded and Embedded Material

Specimens of brain or spinal cord fixed in formalin, Cajal's formol-bromide, or Koenig, Groat and Windle's formalin-acacia can be used to stain oligodendrocytes in frozen, in paraffin, or in celloidin sections. The sections are soaked 3-5 min in 0.02% acetic acid, pH 3.4, then rinsed 2-3 sec in 3% H₂O₂ and transferred to a silver bath prepared as follows: Mix equal parts of 10% AgNO₃ and 10% Na₂WO₄, and dissolve the precipitate with concentrated NH₄OH; avoid an excess of ammonia. Silver at room temperature for 15-20 sec, develop in 1% formalin, dehydrate, and mount. For embedded material, prepare a mixture consisting of 1 part of 10% aqueous Aerosol MA and 4 parts of 10% Aerosol OT in 95% alcohol. Add 5 drops of this mixture to each 50 ml of dilute acetic acid and 3% H₂O₂; 5 drops to each 20 ml of the silver bath.     

A Method to Prevent Wrinkles in Autoradiographic Emulsion when Staining Plastic Embedded Sections with Hematoxylin and Eosin-Phloxine

A procedure was developed which prevents wrinkles in autoradiographic emulsion when sections, embedded in glycol methacrylate, are stained with hematoxylin and eosin-phloxine. Craniofacial tissues labeled with tritiated thymidine were collected and mounted on slides. Slides were dipped in emulsion, stored for one month and developed. The slides were immersed in liquefied celloidin and subsequently stained with a modified hematoxylin and eosin-phloxine procedure. Results showed that the emulsion did not wrinkle and the procedure did not effect the occurrence of labeled cells.     

四氯化碳联合二乙基亚硝胺对小鼠肾脏毒性作用的研究

为了研究四氯化碳(carbon tetrachloride,CCl_4)联合二乙基亚硝胺(diethylnitrosamine,DEN)对小鼠肾脏组织的毒性损伤作用,将30只雄性昆明鼠随机分为对照组和实验组,其中实验组小鼠每周2次腹腔注射15%CCl_4花生油溶液,并且自由饮用0.07%DEN溶液;而对照组小鼠注射等量的纯花生油,自由饮用纯净水,观察并记录各组小鼠的饮水、进食情况和体重变化,以及生活状态的改变。在实验过程中,分别于第3、6、8周解剖两组小鼠各4只,取其肾脏组织样本进行病理方面的组织切片观察。体重统计结果显示,与对照组相比,实验组小鼠体重明显下降。同时,实验组小鼠的肾脏系数明显高于对照组,并且具有显著差异(P0.05)。组织切片观察发现,对照组小鼠肾脏组织结构比较清晰,排列较规则,而实验组小鼠肾脏组织呈现出显著的变性和坏死;且随着处理时间的延长,肾脏组织变得散乱无规则,细胞凝集现象越来越明显,肾小管与集合小管管腔变窄,颗粒样变性。上述结果表明CCl_4联合DEN对小鼠肾脏具有损伤效应,处理时间越长,损伤作用越严重。     

Immunofluorescent Labelling of K-Papovavirus Antigens in Glycol Methacrylate Embedded Material: A Method for Studying Infected Cell Populations by Fluorescence Microscopy and Histological Staining of Adjacent Sections

Trypsin and protease V (pronase) were studied for their ability to enhance immuno-fluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 μm sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.