This user guide provides guidelines and protocols for the proper preparation and pretreatment of fresh, frozen tissues mounted on slides. Fix sections in cold acetone (-20°C) for 2 min. Wipe down the knife holder and anti-roll plate with 100% ethanol. Alternate Protocol. 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. 3. Remove fresh frozen tissue slides from -80°C. 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. Optimal staining is achieved with 5-6 µm thick sections. Frozen Tissue Prep for FISH. 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 . A) RNA isolation from fresh frozen tissue Approx. The Tissue Preparation Guide provides guidance on: Selecting appropriate Visium Spatial slides specific to the Visium Spatial protocol being used. C in PBS before proceeding. 1. This is the second post in a series on immunohistochemistry (IHC). Immerse in 50% EtOH. Embed tissue in OCT before cryosectioning. 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. 2. 2. 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 Fresh Tissue Collection, Processing and Storage . Cut cryostat sections at 5-10 µm and mount on gelatin-coated histological slides. . 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. This is one of a series of protocols on sectioning unembedded plant tissues, prepared by Rosemary White. Preparation of Paraffin Sections and Frozen Tissue for FISH 2006 3 For Frozen sections only A. Pre-fixation. 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. Tumor stiffness measurements can be performed on fresh or snap frozen samples. Fresh Frozen Tissue Preparation Protocol Material preparation: 1. Dehydrate the Tissue 1. tissue elements and the lack of ice crystal artifact. The standard sample weight is 0.5 - 1.0 gram. Fresh tissue freezing - Tissue is in OCT and flash frozen fresh. 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 . Fresh . Temperature is tissue dependent. Frozen serum, plasma & buffy coat (non-viable) are . For frozen tissues, trim to fit the tissue into the plastic cassette and proceed to step #8. 3. Prepare Tissue for Fixed Frozen Sections Materials needed: Fixative ( 2% PFA, 4% PFA, 10% buffered formalin) Cold PBS Liquid nitrogen Dry ice Peel-away base mold OCT (Frozen tissue matrix) Forceps 2. 3. However, volumes and procedures can be adjusted according to the Trizol protocol supplied with each Trizol reagent. Transfer tissue into pre-chilled container on dry-ice. Procedure: Preparing Frozen Sections . RNAscope®Reagent Kits come with a separate RNAscope®Assay User Manual. Prepare 200 mL 50% EtOH, 200 mL 70% EtOH, and 400 mL 100% EtOH. Store in a -70°C freezer until use. 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. 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. Air dry sections for several minutes to remove moisture. Solid tissue biobanks have therefore been established [1]. OCT). 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 . This method allows great structural and biochemical preservation of the tissue and is the format of choice for a majority of downstream analysis. mechanisms directly from tissue samples. All variants identified in the FFPE and fresh frozen tissues are showed in Supplemental Table 1. After you have flash frozen your tissue, you will embed it in OCT. preps. . 3. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies. 2. 2. 5. Protocol on Tissue Preparation and Measurement of Tumor Stiffness in Primary and Metastatic Colorectal Cancer Samples with an Atomic Force Microscope. 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. . Float the can in the liquid nitrogen until the isopentane is cooled. 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 µg DNA. 3. The slides can then be assayed using an RNAscope® Reagent Kit. Tissue/Cells. Dip the slide in 1X TEA buffer. 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. The detection reagents contains AMP 1, APM2, AMP 3, AMP 4, AMP 5, AMP 6, Fast RED-A, Fast RED-B. 4% PFA fixed, sucrose cryoprotected tissue freezing - Tissue is in OCT and may be frozen using dry ice or the flash frozen method. Here we present the detailed protocol for high-throughput proteomic analysis of less than 1 mg fresh-frozen tissue sample using pressure cycling technology . TMPD is added to a subset of the hFresh samples . 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 Dip the slide in nuclease-free water. 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. Standard Protocol. This is the second post in a series on immunohistochemistry (IHC). Common examples include Oil Red O staining for lipids (removed during paraffin processing) and antibodies whose epitopes are masked or destroyed by the ethanols and xylenes and heat involved with paraffin processing. Gently blot excess liquid off of tissue. A standard sample weighs 0.4 - 0.7g on average and is supplied in a standard cryovial or cassette. Cytoplasm and other tissue elements Various shades of pink PROCEDURE NOTES: 1. Dehydrate the Tissue 1. with haematoxylin) if needed We hope you'll have found this protocol a useful start to preparing your frozen IHC sections. '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. After the tissue is frozen, place it in dry ice and move to -80° C until ready for cutting. hFresh represents liver homogenate which was isolated and tested without being frozen. Certain soft tissues, such as brain, are optimally frozen in M-1 medium at −3°C. Both protocols may be widely applied to detect the expression pattern of low abundance mRNAs, such as GalR1. Prepare an isopentane and liquid nitrogen bath. See Troubleshooting. 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. Carry out incubations in a humidified chamber to avoid tissue drying out, which will lead to non-specific binding and high background staining. Tissue-Tek O.C.T. Wear appropriate personal protective equipment to avoid injury and cutaneous absorption. NOTE: use fresh PFA and cool to 4 C before use to avoid increased autofluorescence. Paraffin and frozen sections. RNAlater-ICE solves all of these problems.Simply submerge frozen tissue samples in 10 volumes of RNAlater-ICE and store overnight at -20 or -80ºC (the solution will remain liquid at these temperatures).As the tissue thaws, RNA integrity is protected. Before freezing, the tissue could be dissociated into a single-cell suspension. I saw your protocol, so you do not wait for drying after . Protocol Author Rosemary White Overview This protocol outlines how to section frozen plant tissue using a cryostat. Stain with filtered 0.1% Mayers Hematoxylin (Sigma; MHS-16) for 10 minutes in a 50 ml conical tube. The protocol was adapted from the Puregene Genomic DNA purification kit by Gentra. Do not perform an RNAscope®Assay without the correct user manual. All samples are collected under IRB approval and are prepared in strict accordance with our established . Chill freezer mill with LN2 according to the manufacturer's recommendation. See the 2-step immunofluorescence protocol to be used for fresh frozen tissue sections. The main effect of large ice crystals becomes evident when the fresh-frozen tissue is thawed for various experimental reasons, such as staining for specific molecular markers or histological examination of the tissue structure. Below is a detailed protocol containing explanations and commentary. Frozen Tissue Homogenization Using a Freezer Mill 1. When it comes to frozen embedded block or fresh frozen specimens, use some frozen embedding media to adhere the sample to the mount in the proper cutting . 2. Cells can then be cryopreserved in a suitable freezing medium. Protocol for Fresh Frozen Mouse Brain Tissue Day 1- Slide Prep and Hybridization 1. Fresh frozen tissue is the preferred sample to detect gene mutation due to its superiority in preserving DNA. Tissue may be stored at 4. o. METHODS OF TISSUE FREEZING . Remove the slides from NBF or 4% paraformaldehyde. Do not allow frozen tissue to thaw before cutting. The first post looked at one of the two main ways of carrying out IHC - paraffin embedded sections - and in this post we'll take a look at preparing frozen sections. The following step can be omitted when working . 3. Immediately snap freeze fresh tissue in isopentane mixed with dry ice, and keep at -70 °C. Transport container on dry ice to -80C or liquid nitrogen for long term storage. Cut sections 5-15 μm thick in the cryostat at −20°C. Frozen section guide from Northwestern University and others Frozen sectioning is the method of choice when paraffin processing may interfere with any downstream techniques. (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 37˚C for 30 . 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. Label grinding vials numerically and keep a log of numbers in relation to sample information. Frozen tissues are never allowed to thaw after initial freezing. Best practices for handling tissue samples and Visium Spatial slides before and after cryosectioning. 2-methylbutane (Fisher Scientific, cat#O3551-4). The tissue may be as small as a grain of rice. Part 2: Frozen Tissue Sectioning 1. How It Works The BloodPrep™ chemistry protocol isolates DNA from 150µL (or less) of fresh or frozen human whole blood, up to 106 tissue culture cells, or buccal swab material. 3. All fresh-frozen tissue samples were collected using the same standardized protocol (20)(21) (22) (23), and each frozen section was reviewed by pathologists (P. Micke and J. Botling) to confirm . Dry ice. 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. Immediately immerse the slides in the pre-chilled 10% NBF or 4% PFA. 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. Among 112 patients, 44, 39, 19, 7, 2, and 1 had one, two, three, four, six and seven variants, respectively. To maintain preservation of tissue morphology, do not allow frozen sections to air-dry. We will endeavour to identify this publication when it is published and link it to the. 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. Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. Wear appropriate personal protective equipment to avoid injury and cutaneous absorption. 2. How Our Frozen tissue Is collected and Stored. Dry at room temperature for 15-30 min. 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. 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. If necessary, adjust the temperature of the cutting chamber ±5°C, according to the tissue under study. 3. Enzyme study tissue freezing - Often used for fresh muscle tissue. Place tissue using forceps or spatula into isopentane until completely frozen ~1 min depending upon tissue size. 'Frankenstein' protocol for nuclei isolation from fresh and frozen tissue Customer Developed Protocol COMMUNITY.10XGENOMICS.COM Protocol NOTE:All samples and reagents are kept on ice or at 4 °C (wet ice). We provide automated workflows for labs of all size and budgets, along with a choice selection of extraction methods based on the need. compound (Sakura Finetek from VWR, cat#25608-930). 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. You may also proceed directly to frozen tissue sectioning. FFPE tissue analysis revealed at least one variant in 112 patients (94.9%), yielding a total of 226 variants. Immediately before starting tissue homogenization, using a MagNA Lyser Instrument, 800 µl Lysis Buffer from Incubate the slides for at least 15 MIN at 4°C. Otherwise, a bigger portion of the frozen tissue will experience a freeze- Take an aluminum can cut in half and fill with isopentane. Fill a staining dish with -20°C acetone and place in a -20°C freezer. 2. Fresh tissues represent the tumor . When selecting a protocol for fresh tissue dissociation, we suggest testing two to three dissociation methods, chosen based on tumor type and tissue composition, and processing according to the . Therefore, rapid freezing of fresh tissue is necessary in order to eliminate the formation of larger ice crystals. (A) The Seahorse traces and associated OCR (oxygen consumption rate) quantifications of mouse liver homogenates. Plastic mold (choosing proper size of mold depend on the specimen). This Demonstrated Protocol outlines a method for HMW gDNA extraction from fresh frozen tissue. 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. Cut 5um sections on silanized or positively charged slides. In this video, we outline the process and provide some important tips to help you get the . Remove fresh frozen tissue slides from -80°C. Quickly dissect the tissue, wrap in aluminum foil, and place in the cooled isopentane. Tissue transfers should be performed delicately, with a slotted spoon or reagent scoop. Do not allow frozen tissue to thaw before cutting. Includes required reagents, preparation steps and procedure. Tissues are stored in vapor-phase liquid nitrogen (-190°C). 1. oTransfer tissue to 30% sucrose in a conical tube or screw-cap vial and store at 4 C overnight. One can alternatively start with homogenizing frozen tissue in Trizol reagent and continue with step . 2. As in the contrast isn't as distinguished and there is less nucleoli differentiation. If you prefer a more concise protocol . The following protocol describes the procedure starting with a T175 flask, which should give around 200 µg total RNA at the end. Plastic mold (choosing proper size of mold depend on the specimen). Incubate the slides for at least 15 MIN at 4°C. Optimal staining is achieved with 5-6 µm thick sections. Thaw slides at room temperature and equilibrate in 2X SSC for a couple of min. 4. Freezing and embedding tissue samples prior to cryosectioning. 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. Ying Shen, 1,2 Thomas Schmidt, 2,* and Alba Diz-Muñoz 1,3,4,** . 1. 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. Set cryostat temperature to15°C to 20°C. In the era of precision medicine, there is an increasing need to measure several thousand proteins expressed in minimal amount of fresh-frozen biopsy tissue samples from clinical cohorts. Extraction Services Tissue Embed the tissue completely in OCT compound prior to cryostat sectioning. Respirometry measurements of complex I, II, and IV comparing fresh versus frozen tissue protocols. Wash with PBS and start the Paraffin Pretreatment kit protocol at the .2N HCl step and follow through to the end. Immunohistochemistry on Frozen tissues IHC Protocol - Frozen Tissue: An introduction. Fresh frozen tumour and normal tissue samples are collected from surgical specimens when possible according . This may prevent cracking of the block when sectioning. Pre-chill 200 mL of 10% neutral buffered formalin (NBF) or 4% paraformaldehyde (PFA) in 1X PBS to 4°C. Fresh Frozen Tissue Preparation Protocol Material preparation: 1. Use of forceps may damage tissue integrity. A camel hair brush is useful to help guide the emerging section over the knife blade. Confirmation of the epithelial origin of 2D and 3D cultures was performed using immunohistochemistry (IHC) staining. Adler Lab Protocol H&E (Haematoxylin and Eosin) Staining for Frozen Tissue Sections 1. **If Hamatoxylin is stored in 50 ml tube it should be wrapped in foil. The first post looked at one of the two main ways of carrying out IHC - paraffin embedded sections - and in this post we'll take a look at preparing frozen sections. 8. Place a stainless steel beaker of 2-methylbutane in liquid nitrogen and allow to cool adequately. A fresh frozen method with no OCT matrix. 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. Linked Protocols Making hand sections without support material Using other plant tissues as support tissue to make hand sections Using support… Place in pre-labeled base molds filled with frozen tissue matrix. Mince/chop tissue with a razor blade to small pieces. 2. Rinse in running tap water for 5 minutes Counter stain (e.g. 2-methylbutane (Fisher Scientific, cat#O3551-4). Immunohistochemistry Protocol (Frozen): easy to follow directions describing the step by step experimental procedure. Remove desired tissues, trim and cut tissue no more than 5 mm thick. This manual assay is a single-plex, chromogenic-RED assay. 5 MIN.Wrap frozen blocks with labeled foil, and store at 70°C. Thaw the slide-mounted tissue section to room temperature. Other methods of acceptable frozen section fixation include; Formaldehyde 37-40%, ACS (1089) and Acetone, ACS (10014). 2. However, a clear separation of cytosolic and nuclear fractions from tissues, especially frozen tissues, is challenging. Fix in 4% formaldehyde in PBS at 0 ºC for 15 min. Immunohistochemistry on Frozen tissues IHC Protocol - Frozen Tissue: An introduction. The current state of the art of RNA analysis on tissue Recently the RNAlater was described as protecting RNA from degradation.18-21 None of these approaches offer an acceptable universal means of fixing tissue for the relies almost exclusively on fresh or frozen specimens, preparation of permanent sections and use in histo- limiting its . Immediately immerse the slides in the pre-chilled 10% NBF or 4% PFA. 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. Reagents can be applied manually by pipette, or this protocol can be adapted for automated and semi-automated systems if these are available. Immediately snap freeze fresh tissue in isopentane mixed with dry ice, and keep at -70 °C. Prepare 200 mL 50% EtOH, 200 mL 70% EtOH, and Our clinical repositories have a huge variety of fixed human tissues/ specimens in stock, covering a range of samples that one would expect to find in a clinical tissue bank, particularly for solid tumors. 2. Wash twice with 1X PBS for 5 minutes. Methods for Frozen Tissue Homogenization A. When freezing cells, we recommend starting with at least 1 million total . 3. For tissue stored at -80°C: remove from freezer and equilibrate at -20°C for approximately 15 minutes before attempting to section. All fresh frozen tissue samples are collected under IRB approval by certified medical pathologists. Immerse the slide in cold 4% E.M. grade paraformaldehyde in 1X PBS for 15 minutes. For antibodies made in Rabbits (5-15 µm cryosections on charged (plus) slides) Materials: Acetone Hydrophobic barrier pen Wash Solution Normal Goat Serum Blocking Solution IHC Antibody Diluent 4. Dry ice. No clearing agent. SummaryAutomatic TranslationAugust 25th, 2020. NOTE: use fresh PFA and cool to 4 C before use to avoid increased autofluorescence. It is FRESHLY FROZEN tissue (Gordon Ramsey will kill me for "fresh frozen", ha-ha-ha)))) but yeah, they are essentially unfixed, raw. Cover the entire tissue block with cryo-embedding media (e.g. 2. 1. compound (Sakura Finetek from VWR, cat#25608-930). 3. 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 . Arrange tissue in the matrix near the bottom so tissue is easily exposed when sections are cut. Unfortunately, flash frozen cells or tissue samples generated low-quality data regardless of the ATAC-seq protocol, compared to fresh and cryopreserved samples. We have extensive access to frozen tissue samples in ongoing biorepository collections. 2. 6. The flash frozen tissue is cut at 5 microns. 3. Remove and discard the microtome blade. Answer: If it is not feasible to process fresh tissue, fresh-frozen tissue samples can be used for Single Cell RNA sequencing. Once treated, tissue can be safely stored at 4ºC or even at room temperature (for a limited period of time) and can be further dissected or . Flash frozen tissues are non-fixed post-surgical tissue samples frozen in liquid nitrogen (LN2). 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. Tissue-Tek O.C.T. 4. No separation of the red blood cells is necessary when isolating from whole blood. BloodPrep chemistry is designed to quickly and efficiently purify Dry fixed slides completely (usually 1 hour at room temperature). Optimal cutting temperature (OCT) compound (such as Tissue-Tek; Sakura Finetek USA) .