Part 2: Frozen Tissue Sectioning 1. Transport container on dry ice to -80C or liquid nitrogen for long term storage. Dehydrate the Tissue 1. Fresh tissues represent the tumor . 10 mg fresh frozen sample from HeLa xenograft tissue, were prepared using a scalpel and transferred into a tube of MagNA Lyser Green Beads pre-cooled on dry ice. . The slides can then be assayed using an RNAscope Reagent Kit. The tissue may be as small as a grain of rice. 5. Generally start with 50 to 100 mg fresh or frozen tissue (about the size of a pencil eraser) and expect to get between 20 to 150 g DNA. For tissue stored at -80C: remove from freezer and equilibrate at -20C for approximately 15 minutes before attempting to section. (For freshly harvested, frozen and sectioned slides, can also apply 100 l of 1:200 RNase in 2X SSC to slide under a cover slip and incubate at 37C for 30 . Remove fresh frozen tissue slides from -80C. Otherwise, a bigger portion of the frozen tissue will experience a freeze- Cells can then be cryopreserved in a suitable freezing medium. Fast and Simple Protocols for Mass Spectrometry-Based Proteomics of Small Fresh Frozen Uterine Tissue Sections Abstract Human tissues are an important link between organ-specific spatial molecular information, patient pathology, and patient treatment options. All variants identified in the FFPE and fresh frozen tissues are showed in Supplemental Table 1. Frozen Tissue Prep for FISH. Frozen tissues are never allowed to thaw after initial freezing. 4% PFA fixed, sucrose cryoprotected tissue freezing - Tissue is in OCT and may be frozen using dry ice or the flash frozen method. If there is more tissue, or it is possible to scale up and use a dounce homogenizer in the first step instead of using a . The flash frozen tissue is cut at 5 microns. 1. You may also proceed directly to frozen tissue sectioning. Protocol on Tissue Preparation and Measurement of Tumor Stiffness in Primary and Metastatic Colorectal Cancer Samples with an Atomic Force Microscope. Fresh tissue freezing - Tissue is in OCT and flash frozen fresh. Dip the slide in nuclease-free water. NOTE: use fresh PFA and cool to 4 C before use to avoid increased autouorescence. 'Frankenstein' protocol for nuclei isolation from fresh and frozen tissue for snRNAseq This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, hence the name Frankenstein. Protocol Steps Prepare frozen tissue sections (steps 1-8): Place a freshly dissected tissue block (< 5 mm thick) on to a pre-labeled tissue base mold. Standard Protocol. 2. Certain soft tissues, such as brain, are optimally frozen in M-1 medium at 3C. compound (Sakura Finetek from VWR, cat#25608-930). Temperature is tissue dependent. How It Works The BloodPrep chemistry protocol isolates DNA from 150L (or less) of fresh or frozen human whole blood, up to 106 tissue culture cells, or buccal swab material. Immunohistochemistry Protocol (Frozen): easy to follow directions describing the step by step experimental procedure. 3. To maintain preservation of tissue morphology, do not allow frozen sections to air-dry. Take an aluminum can cut in half and fill with isopentane. Tissue RNAscope 2.5 HD Detection Reagents-RED assay are designed for RNA in situ hybridization (ISH) based on ACD's patented signal amplification and background suppression technology. 3. Embed the tissue completely in OCT compound prior to cryostat sectioning. Incubate the slides for at least 15 MIN at 4C. 3. The Tissue Preparation Guide provides guidance on: Selecting appropriate Visium Spatial slides specific to the Visium Spatial protocol being used. Dehydrate the Tissue 1. 3. Frozen serum, plasma & buffy coat (non-viable) are . Tumor stiffness measurements can be performed on fresh or snap frozen samples. Remove the slides from NBF or 4% paraformaldehyde. Cover the entire tissue block with cryo-embedding media (e.g. For my fresh frozen tissue I'm doing 1 minute 100% alcohol, 1 minute water, 1 minute Mayer's Hematoxylin, 1 minute water 3 times, 1 second Eosin-Y, 10 seconds 100% alcohol. Flash frozen tissues are non-fixed post-surgical tissue samples frozen in liquid nitrogen (LN2). 8. Label grinding vials numerically and keep a log of numbers in relation to sample information. Chill freezer mill with LN2 according to the manufacturer's recommendation. 2. Do not allow frozen tissue to thaw before cutting. Place tissue using forceps or spatula into isopentane until completely frozen ~1 min depending upon tissue size. The standard sample weight is 0.5 - 1.0 gram. Prepare 200 mL 50% EtOH, 200 mL 70% EtOH, and The following step can be omitted when working . Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. Place in pre-labeled base molds filled with frozen tissue matrix. TMPD is added to a subset of the hFresh samples . Set cryostat temperature to15C to 20C. We will endeavour to identify this publication when it is published and link it to the. Prepare an isopentane and liquid nitrogen bath. METHODS OF TISSUE FREEZING . We have extensive access to frozen tissue samples in ongoing biorepository collections. The cross-contamination from frozen tissues is always an issue because of the altered tissue cellular structure caused by the freeze and thaw cycle, improper homogenization, and the use of an inadequate extraction buffer. Incubate the slides for at least 15 MIN at 4C. This may prevent cracking of the block when sectioning. Immunohistochemistry on Frozen tissues IHC Protocol - Frozen Tissue: An introduction. **If Hamatoxylin is stored in 50 ml tube it should be wrapped in foil. Collection Protocol Specifications: a small piece of tissue prepared by a certified medical pathologist is snap-frozen in liquid nitrogen usually within 20 - 30 min after the surgical excision or 4 - 12 hours postmortem in autopsy cases. Best Lab Practices: Whatever is the preferred staining method for frozen section, the following general best lab practices helps to ensure an optimal staining result. Float the can in the liquid nitrogen until the isopentane is cooled. 2. As we noted in our previous post on IHC, with so many steps and possible variations in reagents, tissues and antigens of interest, IHC can be as much art as science. 3. Optimal cutting temperature (OCT) compound (such as Tissue-Tek; Sakura Finetek USA) . Cut cryostat sections at 5-10 m and mount on gelatin-coated histological slides. preps. Respirometry measurements of complex I, II, and IV comparing fresh versus frozen tissue protocols. We provide automated workflows for labs of all size and budgets, along with a choice selection of extraction methods based on the need. Frozen embedding frozen tissue in OCT compound: Embedding frozen tissue is similar as embedding unfrozen tissue, except that you must freeze down the frozen tissue in OCT compound as soon as possible without any delay whenever two of them touch each other. Ensure frozen material remains frozen until samples are mixed with lysis buffer and Proteinase K. Stabilized and fresh tissue should be kept cold or on ice during preparation. Molecular Instruments FRESH/FIXED FROZEN TISSUES Sample preparation protocol 1.Remove frozen sections on slide from -80 C. 2.Fix tissues by immersing slides in ice-cold 4% paraformaldehye (PFA) for 15 min at 4 C. CAUTION: use PFA with extreme care as it is a hazardous material. Protein analysis is demanding in that efficient extraction requires fresh, or fresh-frozen, tissue without fixation, and that no amplification method (such as PCR) exists, so that sufficient material for analysis has to be extracted from the . Tissue may be stored at 4. o. See Troubleshooting. Wash twice with 1X PBS for 5 minutes. This method allows great structural and biochemical preservation of the tissue and is the format of choice for a majority of downstream analysis. How Our Frozen tissue Is collected and Stored. Remove desired tissues, trim and cut tissue no more than 5 mm thick. Protocol Author Rosemary White Overview This protocol outlines how to section frozen plant tissue using a cryostat. 2-methylbutane (Fisher Scientific, cat#O3551-4). Includes required reagents, preparation steps and procedure. Protocol for Fresh Frozen Mouse Brain Tissue Day 1- Slide Prep and Hybridization 1. 6. Dry fixed slides completely (usually 1 hour at room temperature). Fix in 4% formaldehyde in PBS at 0 C for 15 min. Arrange tissue in the matrix near the bottom so tissue is easily exposed when sections are cut. However, a clear separation of cytosolic and nuclear fractions from tissues, especially frozen tissues, is challenging. Immunohistochemistry Protocol for Cryopreservation of Tissues Prior to Fixation This method utilizes frozen tissues that are fixed after snap-freezing and sectioning with a cryostat. Wear appropriate personal protective equipment to avoid injury and cutaneous absorption. Wipe down the knife holder and anti-roll plate with 100% ethanol. This Protocol can produce gDNA with an average size of >200 kb when analyzed on a pulsed-field gel, and typically >80 kb after the Chromium Genome Protocols. 3. Dip the slide in 1X TEA buffer. 4. For cells that are grown on culture plates, carefully remove the cells by manual scraping, trypsinization, or EDTA, and place them into a centrifuge tube. This user guide provides guidelines and protocols for the proper preparation and pretreatment of fresh, frozen tissues mounted on slides. Developed to prepare nuclei isolates from small sample sizes (as little as a grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e . 2. Fix sections in cold acetone (-20C) for 2 min. compound (Sakura Finetek from VWR, cat#25608-930). Optimal staining is achieved with 5-6 m thick sections. . 1. For antibodies made in Rabbits (5-15 m cryosections on charged (plus) slides) Materials: Acetone Hydrophobic barrier pen Wash Solution Normal Goat Serum Blocking Solution IHC Antibody Diluent Reagents can be applied manually by pipette, or this protocol can be adapted for automated and semi-automated systems if these are available. Procedure: Preparing Frozen Sections . 3. Pre-chill 200 mL of 10% neutral buffered formalin (NBF) or 4% paraformaldehyde (PFA) in 1X PBS to 4C. 2. A standard sample weighs 0.4 - 0.7g on average and is supplied in a standard cryovial or cassette. Immerse in 50% EtOH. See the 2-step immunofluorescence protocol to be used for fresh frozen tissue sections. Mince/chop tissue with a razor blade to small pieces. Alternate Protocol. 2. Immerse the slide in cold 4% E.M. grade paraformaldehyde in 1X PBS for 15 minutes. Rinse in running tap water for 5 minutes Counter stain (e.g. Immediately snap freeze fresh tissue in isopentane mixed with dry ice, and keep at -70 C. Note: Depending on local protocols, frozen samples may need to be stored temporarily within the diagnostic laboratory until the pathology report is complete and the samples are released. Answer: If it is not feasible to process fresh tissue, fresh-frozen tissue samples can be used for Single Cell RNA sequencing. Unfortunately, flash frozen cells or tissue samples generated low-quality data regardless of the ATAC-seq protocol, compared to fresh and cryopreserved samples. To characterize and validate this method, we performed a comparative analysis of viability in fresh and viable frozen tissues by evaluating their ability to develop two-dimensional (2D) and three-dimensional (3D; organoid) cultures. No separation of the red blood cells is necessary when isolating from whole blood. This study presents two protocols assaying different mouse brainstem tissue preparations - fresh frozen, or fixed - for simultaneous multiplex fluorescent labelling of mRNA and proteins in situ. Tissue-Tek O.C.T. 2. A camel hair brush is useful to help guide the emerging section over the knife blade. Stain with filtered 0.1% Mayers Hematoxylin (Sigma; MHS-16) for 10 minutes in a 50 ml conical tube. Methods for Frozen Tissue Homogenization A. Before freezing, the tissue could be dissociated into a single-cell suspension. Solid tissue biobanks have therefore been established [1]. SummaryAutomatic TranslationAugust 25th, 2020. The protocol was adapted from the Puregene Genomic DNA purification kit by Gentra. Below is a detailed protocol containing explanations and commentary. Plastic mold (choosing proper size of mold depend on the specimen). Do not allow frozen tissue to thaw before cutting. The detection reagents contains AMP 1, APM2, AMP 3, AMP 4, AMP 5, AMP 6, Fast RED-A, Fast RED-B. However, volumes and procedures can be adjusted according to the Trizol protocol supplied with each Trizol reagent. 3. 2-methylbutane (Fisher Scientific, cat#O3551-4). 2. Frozen Tissue Homogenization Using a Freezer Mill 1. Dry ice. Immediately immerse the slides in the pre-chilled 10% NBF or 4% PFA. Fresh frozen tissue is the preferred sample to detect gene mutation due to its superiority in preserving DNA. Prepare 200 mL 50% EtOH, 200 mL 70% EtOH, and 400 mL 100% EtOH. Quickly dissect the tissue, wrap in aluminum foil, and place in the cooled isopentane. After you have flash frozen your tissue, you will embed it in OCT. This is one of a series of protocols on sectioning unembedded plant tissues, prepared by Rosemary White. All fresh frozen tissue samples are collected under IRB approval by certified medical pathologists. Do not perform an RNAscopeAssay without the correct user manual. 4. Freezing and embedding tissue samples prior to cryosectioning. Wash with PBS and start the Paraffin Pretreatment kit protocol at the .2N HCl step and follow through to the end. Once treated, tissue can be safely stored at 4C or even at room temperature (for a limited period of time) and can be further dissected or . Cut sections 5-15 m thick in the cryostat at 20C. Fresh Tissue Collection, Processing and Storage . Use of forceps may damage tissue integrity. BloodPrep chemistry is designed to quickly and efficiently purify Here we present the detailed protocol for high-throughput proteomic analysis of less than 1 mg fresh-frozen tissue sample using pressure cycling technology . Tissue transfers should be performed delicately, with a slotted spoon or reagent scoop. Tissues are stored in vapor-phase liquid nitrogen (-190C). Thaw the slide-mounted tissue section to room temperature. NOTE: use fresh PFA and cool to 4 C before use to avoid increased autouorescence. RNAlater-ICE solves all of these problems.Simply submerge frozen tissue samples in 10 volumes of RNAlater-ICE and store overnight at -20 or -80C (the solution will remain liquid at these temperatures).As the tissue thaws, RNA integrity is protected. Our tissue/cell workflows allow you to process genomic DNA or total RNA from fresh/frozen tissues or cultured cells, up to 16 samples at once. Draw circles around the tissue with a hydrophobic pen or just dry the slide around the tissue before each incubation step to provide surface tension If your tissue was fresh frozen, fix the tissue for 10 minutes in 75% acetone + 25% ethanol or 4% PFA -> Wash 3-6 x 3 minutes in TBS-T Tissue-Tek O.C.T. Molecular Instruments FRESH/FIXED FROZEN TISSUES Sample preparation protocol 1.Remove frozen sections on slide from -80 C. 2.Fix tissues by immersing slides in ice-cold 4% paraformaldehye (PFA) for 15 min at 4 C. CAUTION: use PFA with extreme care as it is a hazardous material. Fresh frozen tumour and normal tissue samples are collected from surgical specimens when possible according . 3. Frozen section guide from Northwestern University and others Frozen sectioning is the method of choice when paraffin processing may interfere with any downstream techniques. Note: Tissues should be kept moist and cool until snap freezing procedure is started Reagents: Liquid nitrogen 2' Methybutane (Isopentane) OCT embedding compound Plastic embedding mold Forceps Metal cup Container for liquid nitrogen Procedure: 1. One can alternatively start with homogenizing frozen tissue in Trizol reagent and continue with step . A) RNA isolation from fresh frozen tissue Approx. Remove fresh frozen tissue slides from -80C. OCT). tissue elements and the lack of ice crystal artifact. Fill a staining dish with -20C acetone and place in a -20C freezer.