FD Rapid GolgiStain™ Kit
Contents
1. Ram n Moliner and Glaser and Van der Loos This kit has not only dramatically improved and simplified the Golgi Cox technique but has also proven to be extremely reliable and sensitive for demonstrating morphological details of neurons and glia especially dendritic spines The FD Rapid GolgiStain kit has been tested extensively on the brains from several species of animals as well as on the specimens of postmortem human brains for photo samples and references using this kit please visit our web site at www fdneurotech com ll Kit Contents Store at room temperature Solution A 250 ml Solution B 250 ml Solution C 250 ml x 2 Solution D 250 ml Solution E 250 ml Glass Specimen Retriever 2 Natural hair paintbrush 3 Dropping bottle 1 Plastic forceps l User Manual 1 lll Materials Required but Not Included 1 Double distilled or deionized water 2 Plastic glass tubes or vials D WAG RRA 3 Histological supplies and equipment e Gelatin coated microscope slides e Coverslips e Staining jars e Ethanol e Xylene or xylene substitutes e Resinous mounting medium e g Permount e A light microscope Permount is a registered trademark of Fisher Scientific IV Safety and Handling Precautions 1 FD Rapid GolgiStain kit is made for in vitro research use only and not for drug diagnostic or other uses 2 The kit contains reagents that are toxic and harmful in contact with skin or by inhalation and may be fatal if ing2. D TAG RAR Quality amp Excellence Since 1996 FD Rapid GolgiStain Kit A complete Golgi Cox staining system for the study of the morphology of neurons and glia User Manual PK 401 Version 2006 02 FOR IN VITRO RESEARCH USE ONLY not for diagnostic or other uses FD NeuroTechnologies Inc P O Box 785 Ellicott City MD 21041 U S A Phone 410 788 1864 FAX 410 788 1171 www fdneurotech com 2002 2006 FD NeuroTechnologies Consulting amp Services Inc All Rights Reserved D WAG AHA Contents L InitOdHeHol eege 3 II Kit COMMU 10 MEN H 3 III Materials Required but Not Included iiir etta nba rr t oret Rosa uv ecce toni ipis 3 IV Safety and Handling et 4 V Tissue E 4 VI Slain grati ecc ee 6 VU Referentes mr 7 D WAG RHA l Introduction Golgi Cox impregnation has been one of the most effective techniques for studying both the normal and abnormal morphology of neurons as well as glia Using the Golgi technique subtle morphological alterations in neuronal dendrites and dendritic spines have been discovered in the brains of animals treated with drugs as well as in the postmortem brains of patients with neurological diseases However the reliability and time consuming process of Golgi staining have been major obstacles to the widespread application of this technique FD Rapid GolgiStain kit is designed based on the principle of the methods described by
3. Pathological cell cell interactions elicited by a neuropathogenic form of mutant huntingtin contribute to cortical pathogenesis in HD mice Neuron 46 433 444 2005 Ramanan N Shen Y Sarsfield S Lemberger T Sch tz G Linden DJ and Ginty DD SRF mediates activity induced gene expression and synaptic plasticity but not neuronal viability Nature Neuroscience 8 759 767 2005 Moretti P Levenson JM Battaglia F Atkinson R Teague R Antalffy B Armstrong D Arancia O Sweatt JD and Zoghbi HY Learning and memory and synaptic plasticity are impaired in a mouse model of Rett syndrome J Neuroscience 26 319 327 2006 Inan M Lu HC Albright MJ She WC and Crair MC Barrel map development relies on protein kinase A regulatory subunit II B mediated cAMP signaling J Neuroscience 26 4338 4349 2006 Jacobsen JS Wu CC Redwine JM Comery TA Arias R Bowlby M Martone R Morrison JH Pangalos MN Reinhart PH and Bloom FE Early onset behavioral and synaptic deficits in a mouse model of Alzheimer s disease Proc Natl Acad Sci USA 103 5161 5166 2006 For more references using this kit please visit our website at www fdneurotech com Updated 05 31 06
4. a week in the dark Note e To prevent tissue from ice crystal damage and to preserve the best possible cell morphology tissue should be frozen rapidly before sectioning with a cryostat e g by immersing the tissue in isopentane precooled to 70 C with dry ice e The types of cryostat may vary but all types should be able to cut thick sections e g 100 um Contact FD NeuroTechnologies for technical assistance e The 22 C setting of temperature for the cryostat chamber is satisfactory in most cases However variations in type of cryostat and tissue may require a higher or lower chamber temperature in order to cut sections smoothly and without shattering VI Staining Procedure 1 Rinse sections in distilled water 2 times 2 minutes each 2 Place sections in a mixture consisting of 1 part Solution D 1 part Solution E and 2 parts double distilled water for 10 minutes e g Solution D 10 ml Solution E 10 ml Distilled water 20 ml Note e The working solution should be prepared just before use and may be used for up to 100 sections per 100 ml e The bottle and staining jar containing the working solution must be covered to prevent vaporization of the reagent e For the best results the working solution should be stirred frequently during incubation 3 Rinse sections in distilled water 2 times 4 minutes each distilled water should be renewed frequently 4 Counterstain sections with cresyl violet or thionin optional
5. carefully to avoid damage or pressing of the tissue Note e Perfusion of animals and fixation of tissue are not necessary e Large brain specimens should be sliced with a sharp blade into blocks of approximately 10 mm thickness 2 Rinse tissue briefly in double distilled water to remove blood from the surface 3 Immerse tissue in the impregnation solution made by mixing equal volumes of Solutions A and B and store at room temperature for 2 weeks in the dark Replace the impregnation solution after first 6 hours of immersion or on next day A 2 week impregnation time is satisfactory in most cases However variations in type and actual size of tissue may require a shorter or longer duration of impregnation to obtain the best results The ideal time should be obtained by trial for each type of tissue but 3 weeks is often sufficient for most tissues Note that prolonging the impregnation time may cause non specific staining Note e The mixture of Solution A and B should be prepared at least 24 hours prior to use and left unstirred e tis important to use the top part of solution that is free of precipitate e The impregnation solution may be stored at room temperature for up to 1 month in the dark before use e Use at least 5 ml of the impregnation solution for each cubic cm of the tissue to be studied Ge the volume of the impregnation solution should be at least five times that of the tissue Note that use a lesser volume of
6. ested Do not pipette by mouth Avoid inhalation and contact with skin and eyes In case of contact wash immediately with generous amounts of water and seek medical advice If swallowed wash out mouth with water and immediately call a physician 3 Perform experiment under a chemical hood Wear suitable protective clothing gloves and eye face protection while handling kit reagents Wash hands thoroughly after performing the experiment Material safety data sheet MSDS is available at www fdneurotech com V Tissue Preparation e All containers plastic preferred to be used should be cleansed and rinsed with distilled water e Do not use metal implements whenever Solutions A and B are present e Keep containers tightly closed at all times e All tissues treated with Solutions A and B including tissue sections should be protected from light e The following procedure should be performed at room temperature unless specifically indicated Note FD Rapid GolgiStain kit has been proven to produce the best results in animal and postmortem human brain tissues prepared according to the following procedures However it may be used in tissue specimens prepared differently please contact FD NeuroTechnologies for more information before use D WAG RA 1 Experimental animals should be deeply anesthetized before killing The animal brain or postmortem human specimens should be removed from the skull as quickly as possible but handled
7. hl JP Wang Dunlop J Gonzales C Goad MEP Mark RJ and Kwak SP Characterization of the WAVEI knock out mouse implications for CNS development J Neuroscience 23 3343 3352 2003 2 Beggs HE Schahin Reed D Zang K Goebbels S Nave KA Gorski J Jones KR Sretavan D and Reichardt LF FAK deficiency in cells contributing to the basal lamina results in cortical abnormalities resembling congenital muscular dystrophies Neuron 40 501 514 2003 3 Milatovic D Zaja Milatovic S Montine KS Horner PJ and Montine TJ Pharmacologic suppression of neuronal oxidative damage and dendritic degeneration following direct activation of glial innate immunity in mouse cerebrum J Neurochem 87 1518 1526 2003 4 Amateau SK and McCarthy MM Induction of PGE by estradiol mediates developmental masculinization of sex behavior Nature Neuroscience 7 643 650 2004 5 Ishikura N Clever JL Bouzamondo Bernstein E Samayoa E Prusiner SB Huang EJ and DeArmond SJ Notch 1 activation and dendritic atrophy in prion disease Proc Natl Acad Sci USA 102 886 891 2005 D WAG RRA Ren Patterson RF Cochran LW Holmes A Sherrill S Huang SJ Tolliver T Lesch KP Lu B and Murphy DL Loss of brain derived neurotrophic factor gene allele exacerbates brain monoamine deficiencies and increases stress abnormalities of serotonin transporter knockout mice J Neurosci Res 79 756 771 2005 Grillet N Pattyn A Contet C Kieffer BL Goridis C and JF Brunet Generatio
8. impregnation solution may decrease the sensitivity and reliability of staining Warning Solutions A containing potassium dichromate and mercuric chloride and B containing potassium chromate are toxic in contact with skin and may be fatal if swallowed The experiment should be performed under a chemical hood Wear suitable protective clothing gloves and eye face protection while handling the reagents DO NOT POUR THE WASTE OF SOLUTIONS A AND B INTO THE SINK Collect the waste of these solutions in a bottle and call your safety office or a licensed professional waste disposal service to dispose of this material 4 Transfer tissue into Solution C and store at 4 C for at least 48 hours up to 1 week in the dark Replace the solution after first 24 hours of immersion or on next day D WAG RRA 5 Sections up to 240 um thickness may be cut slowly on a cryostat or a similar type of microtome with the chamber temperature set at 22 C Each section should then be transferred with a glass specimen retriever provided and mounted with Solution C on gelatin coated microscope slides a dropping bottle is provided for easy dropping of Solution C onto the slides After absorption of excess solution left on slide with a strip of filter paper sections should be naturally dried at room temperature do not use a fan or hot plate Dried sections should be processed as soon as possible but may be stored in a slide box at room temperature for
9. n and characterization of Rgs4 mutant mice Molecular and Cellular Biology 25 4221 4228 2005 Laub F Lei L Sumiyoshi H Kajimura D Dragomir C Smaldone S Puche AC Petros TJ Mason C Parada LF and Ramirez F Transcription factor KLF7 is important for neuronal morphogenesis in selected regions of the nervous system Molecular and Cellular Biology 25 5699 5711 2005 McLaughlin KJ Baran SE Wright RL and Conrad CD Chronic stress enhances spatial memory in ovariectomized female rats despite CA3 dendritic retraction possible involvement of CA1 neurons Neuroscience 135 1045 1054 2005 Pyter LM Reader BF and Nelson RJ Short photoperiods impair spatial learning and alter hippocampal dendritic morphology in adult male white footed mice peromyscus leucopus J Neuroscience 25 4521 4526 2005 Bj rkblom B Ostman N Hongisto V Komarovski V Fil n JJ Nyman TA Kallunki T Courtney MJ and Coffey ET Constitutively active cytoplasmic c jun N terminal kinase 1 is a dominant regulator of dendritic architecture role of microtubule associated protein 2 as an effector J Neuroscience 25 6350 6361 2005 Niewmierzycka A Mills J St Arnaud R Dedhar S and Reichardt LF Integrin linked kinase deletion from mouse cortex results in cortical lamination defects resembling cobblestone lissencephaly J Neuroscience 25 7022 7031 2005 Gu X Li C Wei W Lo V Gong S Li SH Iwasato T Itohara S Li XJ Mody I Heintz N and Yang XW
10. step D WAG RRA 5 Dehydrate sections in 50 75 and 95 ethanol 4 minutes each do not skip any step 6 Dehydrate sections in absolute ethanol 4 times 4 minutes each do not prolong 7 Clear in xylene or xylene substitutes 3 times 4 minutes each and coverslip in resinous mounting medium e g Permount Note e For the best results use undiluted mounting medium e Golgi stained sections should be protected from light whenever possible Vil References 1 Corsi P Camillo Golgi s morphological approach to neuroanatomy In Masland RL Portera Sanchez A and Toffano G eds Neuroplasticity a new therapeutic tool in the CNS pathology pp 1 7 Berlin Springer 1987 2 Ram n Moliner E The Golgi Cox technique In Nauta WJH and Ebbesson SOE eds Contemporary Methods in Neuroanatomy pp 32 55 New York Springer 1970 3 Graveland GA Williams RS and DiFiglia M Evidence for degenerative and regenerative changes in neostriatal spiny neurons in Huntington s disease Science 227 770 773 1985 4 Robinson TE and Kolb B Persistent structural modification in nucleus accumbens and prefrontal cortex neurons produced by previous experience with amphetamine J Neurosci 17 8491 8497 1997 5 Glaser ME and 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 1981 References using this kit 1 Da
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