Dispense NaOH solution into the cleaned 20-ml scintillation vials. The droplet protocol [5] usually comprises the following steps: EM grid pre-treatment, particle adsorption, washing, and incubation with heavy metal stain. Then place a glass amber bottle on the weigh boat. Clean forceps and eyelash (ethanol and ultrasonication). Make sure to label the vials and store at RT in the “Base” storage container. The basal lamina is labeled with gold particles after immunostaining for laminin. 3. stain solution for TEM, for all of your negative staining applications. It is an amazing substitute for Uranyl Acetate with similar results. Different protocols for negative staining electron microscopy were successfully used [3], [4]. If the amount is not exactly 6.25 g, then adjust the volume of purified water. Filter uranyl acetate and lead citrate with syringe filter. It might be omitted for uranyl acetate staining because the one-step-uranyl-acetate staining also dilutes the salt in the sample. Place this into the staining dish away from the grids, and add ~3 pellets of sodium hydroxide into it. Old batch of 1N NaOH solution can be used for adjusting pH, or otherwise discarded into “Lead -NaOH” waste bottle. Make sure to remove water from both sides of the grid, as well as the slot on wax surface. 1), before dehydration (Berryman and Rodewald 1990). Alcoholic stains work much faster, but can be difficult to control in a reproducible fashion. You should have TEM grids containing your ultrathin sections wedged onto surface of the dental wax. Place a piece of absorbent sheet on the work surface before starting the procedure. Since uranyl acetate is considered radioactive, do all staining in a designated area. Clean freshly cut styrofoam stick with 100% ethanol before cleaning knife. No. Dispense boiled water into the 100-ml glass bottles and label with today's date. Solution will become milky white. c. Cover with foil to protect uranyl acetate stain from light. H�+T�54��32U0 B]cK=sS#ea`��������k���� ��� endstream endobj 10 0 obj 48 endobj 7 0 obj << /Type /XObject /Subtype /Image /Name /im1 /Filter /FlateDecode /Width 318 /Height 106 /BitsPerComponent 8 /ColorSpace [ /Indexed /DeviceRGB 255 6 0 R ] /Length 8 0 R >> stream : QXP009 V 1.0 Title: Uranyl Acetate Staining for Tissue Written by: Ofer Zrihan Approved by:Anya Vainshtein Date of Issue: June 2, 2005 Page 1 of 2 Introduction Uranium is the heaviest metal used in staining and can be used as a general contrast agent. Excess water should be disposed into the "Lead - NaOH" waste bottle. Second step of the protocol. 4. Also make note of any damages to the grid made during staining. It usually dissolves glycogen or renders it unstainable. 2. 100-ml tri-pour plastic beaker × 2; label one as “UA waste” and the other as “lead waste” (They are used to collect UA and lead waste temporarily. This helps to keep old line water out of the boat when filling or leveling. Dispose of these filter papers into "Solid Waste – UA" bag. The efficient and ready to use Uranyless TEM staining solutions is a substitute for TEM staining with toxic uranyl acetate in staining protocol according to Reynolds. Uranyl acetate: Add a drop of uranyl acetate or place grid with sample side on a drop of uranyl acetate (2%, in H 2 O) for 20–60 s. Blot … Place this into the staining dish away from the grids, and add ~3 pellets of sodium hydroxide into it. On the day of sample staining, spin the uranyl acetate solution at 12,000 rpm for 3 min to pellet any unsolubilized uranyl acetate. (2% uranyl acetate should be stored in the fridge away from light.) Clean knife and boat (with purified water and then ethanol) after each cutting session. Cut a series of ultrathin sections. Wipe the sonicator dry and return it to storage. When done, discard in the “Solid Waste – UA” bag. Protocol for Sample Preparation (QX-302) Doc. All items that came in contact with lead nitrate or lead citrate must be rinsed with water into the “lead-NaOH” waste bottle. Draw the saturated aqueous uranyl acetate solution from the middle of the stock bottle into the 5-ml syringe (UA precipitates accumulate at the bottom and liquid surface). UA staining therefore contributes to the stabilization of nucleic acids and confers electron density, staining nucleic acids and certain proteins. UranyLess is an efficient, ready to use staining substitute for uranyl acetate delivering similar results. Then rinse the glassware twice with purified water. Pt-blue was prepared from a reaction of cis-dichlorodiamine-platinum (II) (cis-platin) with thymidine. For cleaning, rinse thoroughly with 70% alcohol --> purified water --> 0.02 N NaOH solution --> purified water, and air-dry completely. Add 4.0 g of NaOH to the beaker and use a small magnetic stirrer to completely dissolve. The TEM grids should be loaded into the pre-made razor slits in the wax bottom of the staining dish in an orderly array. This protocol was adapted from “Ultrastructural Immunochemistry,” Chapter 7, in Immunohistochemistry: Methods Express (ed. ^9�w���=v�׉=l��KV�mf,�ʓ��%Cu�}�{�|�۴!ы� �5��#=�N�VIeݙ6���� �Ns��6�i��^�m�!� =Z�v�Ⲉ��:z"�˽���V,��'Q�ˌ���6� �����;�=��f�h�B��~�xc������D'��~�@>���vG����e5��b��wE��ȊcR�IT��l���RWP����"��XR��Gz"J�O����6}h��n��ER It usually dissolves glycogen or renders it unstainable. It acts as fixative as well as enhancer of contrast during post-staining by interacting with uranyl acetate and lead citrate.It has been indicated that fixation time has an effect on contrast obtained by uranyl acetate contrasting. This protocol is used for staining virus, bacteria, microphage to show their general morphology. J Cell Biol 17:208-212. Before each staining session, re-melt wax and clean wax surface. Material Needed: 0.5% uranyl acetate in distilled water. Ultrathin sections have been stained with an aqueous solution of UAc followed by lead stains. Observe under electron microscope. ��\x� �h��E�>��*Y��Ś�鱜�ֽ�#����u��S�7��ۿ����S��3zP؈z�X�!r��@m�,C����B��ʞW:Q;4YҤ. �h���&´�˹�9p ��"~&��~X���}{3�Nzz�R$z�.��jY�@H�¨�F������!�5ٿ�[�BⰒ$ۭs ɕ�O�?n��� ?���#�ݷ����B���J�9�^.��f�v� �86ME��,x�T-���D�n�R)�S>���j0�Ʃ�ʽ��F��݄���䶁Q��Q=� >���,��4�j����i7��5>�ld /�8��e�� Wick the grids dry using the filter paper wedges. Apply ~300 µl of 0.02 N NaOH solution to moisten the paper so that it will adhere. No. UranyLess is an efficient, ready to use staining substitute for uranyl acetate delivering similar results. If you have not done this in a while, you should ask for a refresher. Then weigh 1.76 g of sodium citrate. Cover the staining dish for now. Place a 55-mm diameter filter paper into the underside of the glass lid cover. Do clean the razor blade with ethanol or high-purity acetone before making slots on wax surface. Dry grids in dust-free location (big glass or plastic Petri dish). Blot the grid on a filter paper before rinsing with room temperature distilled water. Uranyl Acetate (UA) en bloc Protocol. Technical Tip: UranyLess is not air or light sensitive, unlike Uranyl Acetate. Rinse the lid with purified water and wipe dry with Kimwipe. Pour off the rinse water into the waste beaker. If you are using Pioloform-coated grids, Coat fresh grids the day before you plan to use them. Uranyl acetate (UAc) has been generally used as a superb stain for ultrathin sections of plastic-embedded biological materials [ 1–3]. Tannic acid apparently blocks those sites that normally would be contrasted by uranyl acetate or some other staining compounds. This versatile product can be used as a contrast solution both on ultra-fine sections and negative staining. UA-Zero does not contain any radioactive material and is non-toxic. Place the stopper and shake to dissolve completely. Wipe any small spill with a moistened tissue (e.g., Kimwipe). This is used to bring out unit membranes. Place the syringe back onto the rubber stopper. 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In positive staining, uranyl acetate and/or lead citrate are the most commonly used salts. Cover the dish and stain for 5 min. 9 0 obj << /Length 10 0 R /Filter /FlateDecode >> stream Dispose of the uranyl acetate contaminated items, and change the outer layer of gloves. Into the lead citrate mixture, add 8.0 ml of the freshly made 1 N NaOH (aq). This water must be at RT – NEVER add NaOH into hot water. Glassware that came in contact with lead citrate solution should be rinsed first with a small amount of 1N hydrochloric acid solution, then rinsed with RO water (discard into “Lead - NaOH” waste bottle). Use a micropipette with a 200-µl tip to add 100 µl of 1 N NaOH to the vial. Place the lid back onto the staining dish and let the grids air dry in the dish overnight or longer. Under various circumstances, we have tried many different stains and achieved good results. EM Stain 336 Uranyl Acetate Alternative 25ml Average lead time: 30 days. Good clean handling technique from the block trimming steps and forward all contribute to a final clean stain result: Knives with nicks, both Cryotim and Ultra, can add plastic particles and shavings to surface, later causing stains to pool, aggregate, or precipitate. The mono-molecular film of DNA molecules is touched with a 400-mesh copper TEM grid on which a thin (4-8 nm), homogeneous and low grain carbon layer is deposited (see next paragraph). These alternatives to Uranyl Acetate are non radioactive EM grade universal stains. The stain is used just after osmium post-fixation and before dehydration. Confluence Documentation | Web Privacy Policy | Web Accessibility, Harris KM, Perry E, Bourne J, Feinberg M, Ostroff L, and Hurlburt J (2006) Uniform serial sectioning for transmission electron microscopy. Dispense filtered lead citrate solution onto the section side of grids (one drop per grid). It can be used as a direct replacement for Uranyl Acetate with no changes to standard user protocols and delivers high contrast imaging. Stocks. Then, rinse the grids by directing a gentle stream of purified water from squeeze bottle against the side of the interior glass dish, not on the grids directly. The section was fixed by immersion in 2% formaldehyde and embedded in LR White after bulk staining in uranyl acetate. Negative Staining URANYL ACETATE A 1% to 3% solution of uranyl acetate dissolved in distilled water (pH 4.2 to 4.5) can be used to negatively stain many types of samples. Stain grids with uranyl acetate for 15 minutes and lead citrate for 5 minutes. The filtered stain should be stored in the dark at 4˚C and can be used for >1 year. This product is an alternative to the Uranyl Acetate in the classic staining protocol of Reynolds (1963). (. This protocol of double staining has been conventionally used as the most useful and promising staining method [ 4, 5]. Alcoholic, especially, methanolic uranyl acetate is a chemically aggressive solution. Review SDS and Harris Lab SOP for the following hazardous chemicals used in this procedure: Nitric acid: strong oxidizer; corrosive; causes severe skin burns and eye damage; irritant (respiratory), Lead nitrate: oxidizer; toxic (ingestion, inhalation); carcinogen; reproductive toxin; causes serious eye damage; environmental toxin, Sodium hydroxide: corrosive; causes severe skin burns and eye damage; irritant (respiratory), Uranyl acetate: fatal (inhalation, ingestion); flammable. Solid NaOH pellets used during lead citrate staining should be disposed in a designated container (a bottle labeled "NaOH" in NaOH box). Use the "air-gap within tubing" method if peristaltic pump is used to level boat. Carefully tap out about 6.25 g of uranyl acetate powder into the bottle. Otherwise, treat it as non-cantaminated. J Neurosci 26(47):12101-12103. Product Description. Results Contrast The contrast in EM is mainly obtained by electron scattering, as electrons are not absorbed by biological material. Staining: 16. While waiting for the adsorption, take 0.5 ml of 2% uranyl acetate (in water), and dilute in 0.5 ml of water. Store the solution at 4°C in a secondary plastic container. Remove the stopper and add 1.76 g of sodium citrate to the volumetric flask. Use a 5-ml glass pipette to add 4.9 ml of purified water in a 20-ml glass scintillation vial. Pour all waste into “UA-water” waste bottle. Average lead time: 30 days. Does anybody have a protocol for uranyl acetate staining of nanocellulose? Embed in epoxy resin. Or, for critical work (e.g., series cutting), consider using a separate syringe to level boat. Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Cover the dish and stain for 5 min. Introduction. j�)=e�ga�4��9=��Ӌ'�0��4�j�=�Q. Make sure to separate all waste contaminated with UA (including plastic-backed absorbent pad). You must complete required lab safety training before starting this procedure. Dispense filtered uranyl acetate solution onto the section side of grids only (one drop per grid). wide-mouth amber bottle with cap (PTFE-lined), 120 ml capacity, Purified water (double-distilled, ASTM Type I, or equivalent), sonicator (under tissue processing fume hood in 3.360E), concentrated nitric acid (usually 65-70%; Fisher A200). Using a 10-ml borosilicate glass pipette, carefully add 30 ml of the boiled water to the 50-ml volumetric flask. When you have only a small number of grids to stain, use a gentle stream of purified water on the section side of the grid for several seconds. Negative staining with uranyl acetate or phosphotungstic acid (which is a familiar method in the biological research field), staining with OsO 4 or RuO 4, and shadowing with heavy metals such as a Pt-Pd alloy will enhance mass thickness contrast, viz. Make sure to separate all waste contaminated with UA. This paper introduces an aqueous solution of platinum blue (Pt-blue) as an alternative to uranyl acetate (UA) for staining in transmission electron microscopy (TEM). The lead citrate stain was first described by Reynolds (1963). 2% paraformaldehyde in PBS, 4% uranyl acetate (pH 4) and 2% methylcellulose in filtered and deionized water. Rinse using a series of rapid exchanges: a. The stain should be filtered through a 0.22 µm filter that has been pre-rinsed with large volumes of double distilled water. Therefore, an enhanced extraction of cellular materials should be taken into consideration when using methanolic uranyl acetate. Uranyl Acetate binds to nucleic acids, to proteins and to membranous structures. Then discard outer gloves also. Place tip of mPrep/g capsule on Parafilm (Figure D). “Nitric acid” (10% nitric acid and first rinse), “Lead-NaOH” (lead nitrate, sodium citrate, sodium hydroxide, water), “UA-water” (aqueous UA solution and rinse). Uranyl acetate is the acetate salt of uranium; it is a yellow-green crystalline solid made up of rhombic crystals and has a slight acetic odor. Rinse the glassware at least ten times with RO water, with the first rinse collected into the “nitric acid” waste bottle. Paper points are good for this, or long thin triangles of filter paper. UA-Zero ® EM Stain is a patented solution developed as a direct substitute for Uranyl Acetate. Procedure for negative staining of amyloid fibrils for TEM: 1. Glycogen particles, on the other hand, were stained, whereas they are not in non-mordanted sections. The stained and labeled exosomes were analyzed on a Philips™ CM100 100 kV acceleration voltage electron microscope. To observe the immuno-gold labeled EVs under TEM, we processed our samples using the uranyl acetate staining protocol as described below. Close the stock bottle and seal it with a new piece of Parafilm. Uranyl Acetate (UA) en bloc Protocol. Assume room temperature unless otherwise specified. I know that it was used not in replacement, but as a alternative to uranyl acetate in the staining in the ultrathin sections - I think it may apply also to cutaneous tissue staining. A decent spacing between the grids in the wax-filled dish should make it easier to dry them well. If it does not, start over. The current protocol for preparation of samples for electron microscopy in the SEA-PHAGES Laboratory Manual utilizes 1% uranyl acetate as the stain. Place the syringes, hypodermic needle down into the rubber stopper and place all in a 500-ml plastic tri-pour beaker for storage at 4°C. ADD TO BASKET ADD TO QUOTE. e. Dispense into waste. Airdried grids of the sample are viewed under the Transmission Electron Microscope. Bring up the volume to 50 ml with the boiling purified water and mix by inversion. Make a boat with a small (~1/2 in. In a 100-ml beaker, add 30 ml of purified water. The following Personal Protective Equipment is required for this procedure: Nitrile gloves (double-layer required; regularly check for holes), OPTIONAL: plastic apron, shoulder-length gloves, lead glass shield. Dispose of the filter paper adhered to the underside of the glass lid cover into “Solid Waste – No UA” bag. Uranyless is a real alternative to uranyl acetate, without the problems and restrictions, toxicity and waste elimination procedures associated with uranyl acetate. Biochemistry, Molecular Biology, and Cell Biology Protocols >> Negative staining Protocol for Trasmission Electron Microscopy. Wipe exterior of the bottle a moistened tissue (e.g., Kimwipe) twice. 17. Add the 0.02 N NaOH solution as a first rinse to grids (10-15 sec). A modification of the uranyl acetate replacement staining protocol that simplifies the process is described. Does anybody have a protocol for uranyl acetate staining of nanocellulose? Try to use the factory-cut edges if possible. Repeat the water rinses 5 to 10 times. Hazardous Solid Waste: Place all contaminated solid waste (e.g., gloves, pipets, vials, processing dishes, etc.) _� �`��^\K�>�+�1��3�a�{w� Boiled water remaining in the flask can be used to rinse grids after staining. The absorbent sheet on the work surface should be discarded in "Solid Waste – UA" bag. Monitor the area for radioactivity, as described in the Harris Lab SOP for uranyl acetate. Please read the Material Safety and Data Sheets and product description of all materials that you will be using before using any of those materials, and follow appropriate laboratory safety guidelines. × 2 in.) Measure out purified water in a graduated cylinder and add to the bottle. 2. Close it with a cap and gently shake to mix. All items that came in contact with UA must be rinsed with water into the “UA-water” waste bottle and disposed of in the “Solid Waste – UA” bag. Hazardous Liquid Waste: Pour all waste into the proper waste collection bottles available on bench top or in the corrosive cabinet in NHB 3.360E. Protocol for Sample Preparation (QX-102) Doc. This is used to bring out unit membranes. Dilute concentrated nitric acid in a 1000-ml Erlenmeyer flask (or Fleaker) for the final concentration of 10%. Waiting until the next day will markedly reduce the quality of the stain. &ۥ���}ҵI6�"�G΂emy1�2�U�~?�E�%��`�N1]hc{U Pour off the stain into a 100-ml tri-pour plastic beaker labeled “UA waste”. Transfer the NaOH solution from the beaker into another 50-ml volumetric flask. Consequently, the highest possible contrast is given by saturated methanolic uranyl acetate stain. Since the introduction of heavy metal salts as versatile staining reagents for transmission electron microscopy of biological materials [1, 2], uranyl acetate has been extensively used as a superb stain not only for thin sections of plastic-embedded tissues and cells, but also for high-contrast negative stains for biological macromolecules [3, 4]. Keep blocks free of dust when trimming with air gun. We can offer three alternatives to Uranyl Acetate for territories where shipping of radioactive substances is prohibitive. Add about 750 ml of purified water in the 1000-ml Erlenmeyer flask (or Fleaker) and heat to rapid boil on hotplate. : QXP004 V 1.0 Title: Mild Uranyl Acetate Staining for Cells Written by: Ofer Zrihan Approved by: Ilit Leizerman Date of Issue: June 20, 2005 Page 1 of 1 Introduction Uranium is the heaviest metal used in staining and can be used as a general contrast agent. Place the stopper and mix by inversion. Pour all liquid waste into "Lead citrate-NaOH" bottle. Collect the sections on PEI-coated grids. Vials must be uncapped. This procedure involves the use of hot plate. While this compound provides excellent contrast for phage samples for transmission electron microscopy, uranyl acetate poses a number of Filter with 0.2um syringe filter. Dehydrate through a series of ethanols or acetones and propylene oxide. Use Parafilm to seal when the bottle is just cool enough to handle comfortably. Dispose of the uranyl acetate contaminated items, and change the outer layer of gloves. After several min, pour 10% nitric acid into all other glassware (i.e., 50-ml volumetric flasks, 100-ml glass beakers, 20-ml borosilicate scintillation vials, 100-ml Kimax glass bottles, stirrer) and soak for additional several min. The stain is used just after osmium post-fixation and before dehydration. Label the beaker with today's date. https://labtech-em.com/em/uranyless-tem-staining-alternative-uranyl-acetate Wipe any spill with a moistened Kimwipe. Before starting, even if you have done this procedure before, review relevant Safety Data Sheets and Harris Lab SOP (also see below), ensure you have all reagents and supplies listed below, ensure all equipment is in good working order, have all waste containers ready (also see Clean-up), plan your schedule well so that you wouldn’t have to rush. 100 ml water for 6.25 g UA (i.e., 16 ml water per 1 g UA). AVOID getting any stain on the 'back side of the grid' where it is very hard to clean due to the depth of the Synaptek grid. Drying as much as possible the wax slots used to hold the grids may reduce contamination. Use the filter paper wedges to wick the grids dry of water. Avoid getting the backside wet, but if you do, then be sure to rinse and dry both sides between stains. Final cooling should be done in a sealed bottle. b. Cover the staining dish for now. Otherwise the film will trampoline and cause sections to have 'pockets' where stain can collect. *[v���%��^ƛ��M&�pb�;z̡�Qz��D���٧'=�C�V˗w7��_)��`�J�wZ+jS%l�#��ҧ>�^�'|[o�ύ5L�fG� Maintain rapid boiling for at least 30 min to de-gas CO. Spin down at 4 degrees C for 5 minutes. Make two chambers putting the lid of a petri dish on the parafilm. Use the same beaker to dispense purified water and bring the volume to 50 ml. Pour off the lead stain into the lead waste beaker. Reagents Required. Uranyl acetate, which is used as a general stain, can be specific to nucleic acids when used under certain conditions. Preparing the Grids. Apply rinses with purified water 5 to 10 times, in the same manner as done for uranyl acetate above. Uranyl Acetate binds to nucleic acids, to proteins and to membranous structures. Pour this into the lead waste beaker. Add 143 ml of nitric acid into 857 ml of RO water and mix completely. Discard all contaminated items into "Solid Waste – no UA" bag. Place a large plastic weigh boat on a scale in the fume hood. Let it dry. Prepare a solution of 2% uranyl acetate in DDI water. If water in the sonicator gets contaminated with UA, discard into the "UA-water" waste bottle. Washing which is a very critical process of the stain is done to eliminate excess salts from outside the stain. I am looking for a protocol since the lack of negative staining in compromising most of my recent TEM pictures. Pour all waste into “UA-water” waste bottle. d. Hold for desired staining time, typically 3–15 minutes. £90.69 . Make sure this dish is absolutely clean and dry before and after use, keep it covered when not in use. Then discard the solution into “nitric acid” waste bottle. Cut off two big pieces of parafilm and attach it to the lab bench by pressing down the periphery of the parafilm. Make sure to record the grid storage location (i.e., grid box ID and position in the box). 2% aqueous phosphotungstic acid (adjust pH to 7.3 using 1N NaOH). Glass Petri dish set (60-mm diameter; dish plus the lid), containing about 4 mm of hardened dental wax melted in the bottom dish. %PDF-1.2 %���� Place the grids into slots of wax surface. The Parafilm boat should be disposed in a solid waste bag. For rinse after lead citrate staining, use purified water that was boiled and cooled right before rinsing. Use clean boat water for sectioning (filtered through a syringe-filter unit). Dispose of the weigh boat and any contaminated items (incl. around the tightened cap of the bottle. Set aside to air dry overnight (or longer). pH 5.15 Maleate buffer: 11.6 g maleate/500 ml dH2O, adjusted to pH 5.15 with NaOH
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