Hito Golgi-Cox OptimStain™ Kit
Contents
1. HITO Biotec in situ detection Hito Golgi Cox OptimStain Kit An easy to use Golgi Cox staining system for the morphological characterization of the neurons and glia cells User Manual and Material Safety Data Sheet FOR IN VITRO RESEARCH USE ONLY Hitobiotec Inc a fe H ITO Simple solution for your research Biotec in situ deiection Hito Golgi Cox OptimStain Kit An easy to use Golgi Cox staining system for the morphological characterization of the neurons and glia cells User Manual and Material Safety Data Sheet FOR IN VITRO RESEARCH USE ONLY Hitobiotec Inc 2012 All Rights Reserved VI VII VIII Index Introduction Kit Contents Impregnation Solution Preparation Tissue Preparation Standard Protocol Tissue Preparation Vibratome Protocol Staining Procedure Alternative Protocol for Microtome References Material safety data sheet MSDS o 0 N oa A WO N 11 12 I Introduction Golgi Cox impregnation or Golgi staining has been recog nized as one of the most elegant and effective procedures for studying the morphology of neurons as well as glia In recent years the Golgi Cox staining method remains as a primary technique for visualization of the dendritic branching pattern and dendritic spines because it allows isolation and visualization of the dendritic arbours from a minor random fraction of the neurons in a certain brain area Accordingly Golgi techniques are n2. 10 mm thickness Rinse tissue in double distilled water for 2 3 seconds to remove blood from the surface Transfer tissue into the impregnation solution that is at least five times the volume of the tissue and store at room temperature in the dark Replace the impregnation solution on next day after 12 24 hours and store at room temperature 20 25 C for two weeks in the dark To avoid non specific stain ing do not extend the impregnation time Transfer tissue into Solution 3 that is at least five times the volume of the tissue Store at 4 C in the dark Replace Solution 3 after 12 hours and continue to store at 4 C in the dark for 24 to 72 hours Place 300 to 500 ml isopentane in a metal container large enough to hold a corresponding sieve like basket Place the metal container with the isopentane in dry ice for 15 to 30 min until the temperature of the isopentane reaches 70 C Place the tissue on the mesh bottom of the sieve like basket in a manner that preserves the normal shape of the tissue Slowly immerse the basket with the tissue in the cooled isopentane for 30 sec to 1 min The time of immersion is absolutely critical it must be long enough to result in complete freezing of the tissue but not so long that the tissue cracks lt may be necessary to test various times to determine the optimal time 10 11 12 13 14 15 16 17 Rapidly remove the basket with the frozen tissue
3. Dehydrate slides in 100 ethanol 3 times 3 minutes each Clear in xylene 3 times 4 minutes each and apply coverslip over sections using undiluted xylene based resinous mounting medium Allow to dry The slides can be viewed after drying by bright field microscopy VIIL References 1 Ramon Moliner E A tungstate modification of the Golgi Cox method Stain Technol 33 19 29 1958 2 Ramon Moliner E Vane M A amp Fletcher G V Basic Dye Counterstaining of Sections Impregnated by the Golgi Cox Method Stain Technol 39 65 70 1964 3 Pilati N Barker M Panteleimonitis S Donga R amp Hamann M A rapid method combining Golgi and Nissl staining to study neuronal morphology and cytoarchitecture J Histochem Cytochem 56 539 550 doi jhc 2008 950246 pii 10 1369 jhc 2008 950246 2008 4 Rosoklija G et al Optimization of Golgi methods for impregnation of brain tissue from humans and monkeys J Neurosci Methods 131 1 7 doi S0165027003001 997 pii 2003 5 Spacek J Dynamics of the Golgi method a time lapse study of the early stages of impregnation in single sections J Neurocytol 18 27 38 1989 6 Gibb R 8 Kolb B A method for vibratome sectioning of Golgi Cox stained whole rat brain J Neurosci Methods 79 1 4 doi S0165027097001635 pii 1998 7 Gomez Villalobos M J Gordillo A C Lopez J R amp Flores G The utility of the Golgi Cox method in the morphological characte
4. times that of the tissue e g 5 ml or more of the impregnation solution for 1 cm of the tissue Warning The kit contains reagents that are toxic and harmful in case of ingestion inhalation skin or eye contact Perform experiment under a chemical hood and wear protective clothing gloves goggles face shield or safety glasses while handling kit reagents Wash hands thoroughly with soap and water after handling IF SWALLOWED Rinse mouth with water and immedi ately call a doctor or Poison Control Center IF ON SKIN OR IN EYES Wash immediately with plenty of water and seek medical advice IF INHALED Move person to fresh air and call a doctor or Poison Control Center for further treatment advice Never pour the waste of Solution 1 and 2 into the sink Collect the waste solutions in a tightly closed glass or HDPE container and call your safety administration or a licensed professional waste disposal service to dispose of this material For more information please read the MSDS 4 IV Tissue Preparation Standard Protocol 1 Prepare animal for infusion by administering a lethal dose of anesthesia Monitor it until the point when the animal fails to respond to pinching of the foot Do not perfuse with buffer or fixative Remove the tissue brain spinal cord or heart as soon as possible Handle with care and avoid damage of the tissue Large specimens should be sliced with a sharp blade into blocks of approximately
5. ATING HEALTH 4 FLAMMABILITY O REACTIVITY 3 Potential Health Effects Inhalation May be fatal if inhaled Material is extremely destructive to the tissue of the mucous membranes and upper respiratory tract Skin Causes skin burns skin irritation May be fatal if absorbed through skin Eyes Causes eye burns eye irritation Ingestion Toxic if swallowed May be fatal if swallowed Causes burns 4 FIRST AID MEASURES General advice Consult a physician Show this safety data sheet to the doctor in attendance Move out of dangerous area If inhaled If breathed in move person into fresh air If not breathing give artificial respiration In case of skin contact Take off contaminated clothing and shoes immediately Wash off with soap and plenty of water Consult a physician In case of eye contact Continue rinsing eyes during transport to hospital Rinse thoroughly with plenty of water for at least 15 minutes and consult a physician If swallowed Do NOT induce vomiting Never give anything by mouth to an unconscious per son Rinse mouth with water Consult a physician 5 FIRE FIGHTING MEASURES Flammable properties Flash point no data available Ignition temperature no data available Suitable extinguishing media Use water spray alcohol resistant form dry chemical or carbon dioxide Special protective equipment for fire fighters Wear self contained breathing apparatus for fire fighting if necessary 13 6 ACCID
6. Causes eye burns eye irritation Toxic if swallowed May be fatal if swallowed Causes burns 15 12 ECOLOGICAL INFORMATION Elimination information persistence and degradability Refer to component MSDS Ecotoxicity effects Refer to component MSDS Further information on ecology Refer to component MSDS 13 DISPOSAL CONSIDERATIONS Product Observe all federal state and local environmental regulations Contact a licensed professional waste disposal service to dispose of this material Contaminated packaging Dispose of as unused product 14 TRANSPORT INFORMATION DOT US UN Number 3316 Class 9 Packing Group III Proper shipping name Chemical kits Reportable Quantity RQ 2000 Ibs IMDG UN Number 3316 Class 9 Packing Group III Proper shipping name Chemical kits Reportable Quantity RQ 2000 Ibs IATA UN Number 3316 Class 9 Packing Group III Proper shipping name Chemical kits Reportable Quantity RQ 2000 Ibs 15 OTHER INFORMATION Further information The above information is believed to be correct but does not purport to be all inclusive and shall be used only as a guide The information in this document is based on the present state of our knowledge and is applicable to the product with regard to appropriate safety precautions It does not represent any guarantee of the properties of the product Hito biotech Inc shall not be held liable for any damage resulting from handling or from
7. ENTAL RELEASE MEASURES Personal precautions Use personal protective equipment Avoid dust formation Avoid breathing dust Ensure adequate ventilation Evacuate personnel to safe areas Environmental precautions Prevent further leakage or spillage if safe to do so Do not let product enter drains Discharge into the environment must be avoided Methods for cleaning up Pick up and arrange disposal without creating dust Keep in suitable closed con tainers for disposal 7 HANDLING AND STORAGE Handling Perform experiment in a properly functioning chemical hood which is vented to the outside Wear glasses and disposable gloves while handling kit reagents Wash hands thoroughly after performing the test Storage Keep container tightly closed in a dry and well ventilated place Store at room temperature preferably in a cool place 8 EXPOSURE CONTROLS PERSONAL PROTECTION Contains no substances with occupational exposure limit values Personal protective equipment Respiratory protection Where risk assessment shows air purifying respirators are appropriate use a full face respirator with multipurpose combination US or type ABEK EN 14387 respirator cartridges as a backup to engineering controls If the respirator is the sole means of protection use a full face supplied air respirator Use respirators and components tested and approved under appropriate government standards such as NIOSH US or CEN EU Hand protect
8. contact with the above product See reverse side of invoice or packing slip for additional terms and conditions of sale 16 Notes Notes Hitobiotec Inc P O Box 7671 Wilmington DE 19803 U S A Phone 302 385 6188 Email info O hitobiotec com www hitobiotec com
9. from isopentane detach the tissue from the mesh and place it briefly on absorbent paper in dry ice box to remove excess isopentane Wrap the dried frozen tissue in aluminum foil and store at 70 C until sectioning is performed Set the cryostat chamber temperature at 19 C Place specimen holder cryostat chuck on dry ice and add embedding matrix or distilled water on the surface of the specimen holder chuck As the embedding matrix or water begins to freeze place the frozen tissue into it so that the tissue adheres to the specimen holder chuck Pour embedding matrix over the frozen tissue to provide a thin coat that aids in maintaining the integrity of the tissue sections during cutting Slowly cut the tissue into sections 80 200 pum thickness on a cryostat with the chamber temperature set at 19 C Add a few drops of Solution 3 to a gelatin coated slide with a dropping bottle Using a Glass Specimen Transfer Tool or Fine Tip Natural Hair Brush provided in the kit transfer sections from the specimen holder chuck to a gelatin coated slide Using the edge of a filter paper strip remove excess Solution 3 Air dry slides over night at room tempera ture in the dark Dried sections should be processed as soon as possible but may be stored at room temperature for up to three days in the dark Note The 19 C setting is satisfactory in most cases but may need optimization for different cryostat and tissue ty
10. ion Handle with gloves Eye protection Safety glasses with side shields conforming to EN166 Hygiene measures Avoid contact with skin eyes and clothing Wash hands before breaks and imme diately after handling the product 9 PHYSICAL AND CHEMICAL PROPERTIES 14 Appearance Form Safety data pH Melting point Boiling point Flash point Ignition temperature Lower explosion limit Upper explosion limit Water solubility liquid no data available no data available no data available no data available no data available no data available no data available no data available 10 STABILITY AND REACTIVITY Storage stability Stable under recommended storage conditions Avoid Light Materials to avoid Strong oxidizing agents metals Hazardous decomposition products Hazardous decomposition products formed under fire conditions Hydrogen chloride gas Mercury mercury oxides Potassium oxides Chromium oxides 11 TOXICOLOGICAL INFORMATION Acute toxicity Irritation and corrosion Sensitisation Refer to component MSDS Refer to component MSDS Refer to component MSDS Signs and Symptoms of Exposure no data available Potential Health Effects Inhalation Skin Eyes Ingestion May be fatal if inhaled Material is extremely destruc tive to the tissue of the mucous membranes and upper respiratory tract Causes skin burns skin irritation May be fatal if ab sorbed through skin
11. ml 120 ml Solution 4 250 ml 125ml 60ml Solution 5 250 ml 125ml 60ml Dropping Bottle 20 ml 1 1 1 Staining Jar 25 ml 2 1 1 Fine Tip Natural Hair Brush 2 2 1 Glass Specimen Transfer Tool 2 2 1 Hito Dual Safe Gelatin coated 1 1 0 Slide Sample User Manual and MSDS Note Before using Hito Golgi Cox OptimStain Kit please make sure you have the following Required Equipment Materi als in your lab not included in the kit 1 Cryostat capable of cutting 80 to 200 um thick sections at 19 C or vibratome 2 Dry ice O C T compound isopentane ethanol xylene double distilled or deionized water 3 Plastic glass tubes or vials 4 Gelatin coated slides recommend Hito Dual Safe Gelatin coated Slide Cat HTHS0102 and coverslips 5 Staining jars for slides wash 6 Resinous mounting medium 7 Light microscope Impregnation Solution Preparation Clean all containers then rinse with distilled water Do not use metal instruments Mix an equal volume of Solution 1 and 2 e g mix one part Solution 1 and one part Solution 2 in a clean glass or plastic container Keep the container tightly closed Do not stir the solution mixture Store at room temperature in the dark e g wrapped with aluminum foil for at least 24 hours before use and for use within one month Using the supernatant of the mixed solution precipitate free for the impregnation The volume of the impregnation solution should be at least five
12. ot only useful for pure anatomical studies but are also widely used in studies examining behavioral morphological relationships 36 In some studies the Golgi Cox method has been used to identify and study the autonomic innervations in the heart and the results show that the Golgi Cox method is an attainable and useful tool to identify and study the morphological characteristics of the autonomic innervations in peripheral tissues However the Golgi Cox staining procedure is time consuming the yield of stained cells is usually low and the results are often unreliable Hito Golgi Cox OptimStain Kit offers a simple solution to these problems Designed based on the methods described by Glaser and Van der Loos this kit makes dramatic improvement of the Golgi Cox technique The procedures are simplified and the processing time is greatly reduced This kit delivers stable and improved staining quality with minimal overstains and artifacts when used properly Hito Golgi Cox OptimStain Kit has been tested on the brains spinal cords and hearts from several species of animals and proven to be sensitive for demonstrating morphological details of neurons and glia For photo samples please visit our web site at www hitobiotec com Il Kit Contents Store Hito Golgi Cox OptimStain Kit at room temperature Kit Contents Standard Kit Small Kit Mini Kit Solution 1 250 ml 125ml 60ml Solution 2 250 ml 125 ml 60ml Solution 3 500 ml 250
13. otome VII Alternative Protocol for Microtome Prepare animal for infusion by administering a lethal dose of anesthesia Monitor it until the point when the animal fails to respond to pinching of the foot Do not perfuse with buffer or fixative Remove the tissue brain spinal cord or heart as soon as possible Handle with care and avoid damage of the tissue Large specimens should be sliced with a sharp blade into blocks of approximately 10 mm thickness Rinse tissue in double distilled water for 2 3 seconds to remove blood from the surface Transfer tissue into the mixed impregnation solution that is at least five times the volume of the tissue and store at room temperature in the dark Replace the impregnation solution on next day after 12 24 hours and store at room temperature 20 25 C for two weeks in the dark To avoid non specific stain ing do not extend the impregnation time Transfer tissue into Solution 3 that is at least five times the volume of the tissue Store at 4 C in the dark Replace Solution 3 after 24 hours and continue to store at 4 C in the dark for 5 7 days After 5 7 days processing in Solution 3 transfer the tissue into a new vial and wash the tissue with running tap water over night at room temperature After water rinse dehydrate the tissue in a graded increasing concentrations ethanol water series at room temperature clear tissue in Chloroform for 12 24 hours and embed tis
14. pes in order to cut sections smoothly and maintain integrity of the sections Don t store the sections for a long time black crystal line background will appear V Tissue Preparation Vibratome Protocol Prepare animal for infusion by administering a lethal dose of anesthesia Monitor it until the point when the animal fails to respond to pinching of the foot Do not perfuse with buffer or fixative Remove the tissue brain spinal cord or heart as soon as possible Handle with care and avoid damage of the tissue Large specimens should be sliced with a sharp blade into blocks of approximately 10 mm thickness Rinse tissue in double distilled water for 2 3 seconds to remove blood from the surface Transfer tissue into the impregnation solution that is at least five times the volume of the tissue and store at room temperature in the dark Replace the impregnation solution on next day after 12 24 hours and store at room temperature 20 25 C for two weeks in the dark To avoid non specific staining do not extend the impregnation time Transfer tissue into Solution 3 that is at least five times the volume of the tissue Store at 4 C in the dark Replace Solution 3 after 12 hours and continue to store at 4 C in the dark for 24 to 72 hours Embed the tissue in low gelling temperature agarose Cut vibratome sections at 80 200 um thickness into double distilled water Using a Fine Tip Natural Hair Brush pro
15. rization of the autonomic innervation in the rat heart J Neurosci Methods 179 40 44 doi S0165 0270 09 00037 5 pii 10 1016 j neumeth 2009 01 004 2009 8 Glaser E M amp Van der Loos H Analysis of thick brain sections by obverse reverse computer microscopy application of a new high clarity Golgi Nissl stain J Neurosci Methods 4 117 125 doi 0165 0270 81 90045 5 pi 1981 IX Material Safety Data Sheet MSDS Date Updated 8 22 2012 Version 1 9 1 Product and Company Information Product Name Hito Golgi Cox OptimStain Kit Product Number HTKNS1125 Brand Hitobiotec Company Address Hitobiotec Inc P O Box 7671 Wilmington DE 19803 USA Technical Phone 302 385 6188 Emergency Phone 302 385 6188 2 Composition and Information on Ingredient Substance Name CAS SARA 313 Hito Golgi Cox OptimStain Kit None No Ingredient Name CAS SARA 313 WATER 7732 18 5 No PROPRIETARY COMPONENT S None No Mercury II chloride 7487 94 7 Yes Potassium chromate 7789 00 6 Yes Potassium dichromate 7778 50 9 Yes 3 Hazards Identification EMERGENCY OVERVIEW Toxic Dangerous for the environment Harmful by inhalation or in contact with skin or eyes May be fatal if swallowed or absorbed through skin Possible risk of irreversible damage to skin mucous membranes eyes blood kidneys and diges tive respiratory reproductive and central nervous systems 12 HMIS RATING HEALTH 4 FLAMMABILITY O REACTIVITY 0 NFPA R
16. sues in paraffin blocks recommend paraffin with a lower melting point Turn on the water bath and set the temperature at 45 C Use fresh deionized water Insert the paraffin block into the microtome chuck Set the dial to cut 50 80 um sections Cut sections very slowly and pick them up with forceps or a fine paint brush and float them on the surface of the water bath Float the sections onto the surface of gelatin coated slides 10 11 12 13 14 15 16 17 18 19 20 21 Drain slides upright and dry at 37 C for a minimum of 15 minutes Place the slides with paraffin sections in a 60 C oven for 2 hours preferably overnight to bond the tissue to the glass Slides can be stored in a slide box at room temperature Deparaffinize sections in xylene 3 times 5 minutes each Rehydrate sections in 100 95 70 and 50 etha nol 2 times 5 minutes each Rinse slides in distilled water 3 times 3 minutes each Mix 5 ml Solution 4 5 ml Solution 5 and 15 ml double distilled water in a 25 ml staining jar provided in the kit then place slides in the solution mixture Tightly close the staining jar and wait for 10 minutes Rinse slides in distilled water 2 times 3 minutes each distilled water should be renewed frequently Counterstain sections with cresyl violet optional step Dehydrate slides which was previously rinsed with distilled water in 50 75 and 95 ethanol 4 minutes each
17. vided in the kit mount the floating sections onto gelatin coated slides Add a few drops of Solution 3 to mounted sections with a dropping bottle wait for 1 2 minutes using the edge of a filter paper strip remove excess Solution 3 Air dry slides over night at room temperature in the dark Dried sections should be processed as soon as possible but may be stored at room temperature for up to three days in the dark VI Staining Procedure Rinse slides in distilled water 2 times 3 minutes each Mix 5 ml Solution 4 5 ml Solution 5 and 15 ml double distilled water in a 25 ml staining jar provided in the kit then place slides in the solution mixture Tightly close the staining jar and wait for 10 minutes Rinse slides in distilled water 2 times 3 minutes each distilled water should be renewed frequently Counterstain sections with cresyl violet optional step Dehydrate slides which was previously rinsed with distilled water in 50 75 and 95 ethanol 4 minutes each Dehydrate slides in 100 ethanol 2 times 3 minutes each Clear in xylene 2 times 4 minutes each and apply coverslip over sections using undiluted xylene based resinous mounting medium Allow to dry The slide can be viewed after drying by bright field microscopy Note If you do not have a cryostat processed tissue can be prepared as paraffin sections with microtome as an alternative option see VI Alternative Protocol for Micr
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