ATFM 2015 ATFM Sample preparation guide

Transcription

1 ATFM 2016 Sample preparation guide The workshop is organized in the framework of the Czech-BioImaging research infrastructure supported by MEYS (LM ) 1

2 Table of Contents General recommendations... 3 Basic protocols to treat coverslip and mounting media preparation... 4 Special requirements for each method:... 5 FRET / FLIM... 5 SIM... 5 STED... 6 FRAP

3 General recommendations Slides Standard 76x26x1 mm slides. Coverslips All microscopes which will be used for practical ATFM course are equipped with objectives dedicated for #1.5 coverslip thickness (170 µm). For superresolution methods - SIM and STED - the high precision coverslips are recommended (170 ± 5 µm). No additional coating is needed. We recommend to increase hydrophilic character of the coverslip surface by the basic treatment (Tab1). Any additional surface modification/coating could possibly influence superresolution methods, thus if you are planning to modify the coverslip surface and are interested in superresolution, please, contact us to more detailed discussion/recommendation. Dishes for live cells For FRAP and TIRF experiments we highly recommend 3.5mm petri dish with glass-bottom, #1.5 thickness (170 µm), InVitro Scientific, Mattek or Ibidi. We also can deal with Lab Tek II coverglass system. HCM - we can work with all variety of petri dishes or well-plates; glass bottom is recommended for high NA oil objectives, depends on requirements. Live cells handling Our facility is equipped with 37 C, 5% CO 2 incubator for live cells, if you need other cultivation temperature, let us know and we will try to organize an alternative. In our facility, you find a laminar flow box for sterile handling with cell cultures. Microscopes for FRAP, TIRF and HCM are equipped with environmental chamber (37 C, 5% CO 2 ). If you need other temperature for your live cell imaging, let us know in advance to organize participants into groups according to requirements. 3

4 Media for live cells For the live cell imaging (mostly for TIRF, FRAP), we recommend media without phenol red; we will provide you with excellent FluoroBrite medium for live cell microscopy. If you have any special requirements for the medium, bring with you any additive you need or bring your own microscopic medium. Basic protocols to treat coverslip and mounting media preparation Tab1: Basic coverslips treatment to increase the hydrophilic character of the glass surface: Make 1M HCl in a glass dish and put coverslips in it. Incubate coverslips in HCl (agitation on orbital shaker ~200rpm) for at least 4 hours (RT is sufficient, C is optimal). Wash the coverslips extensively in distilled water to ph 7. Rinse coverslips in 96% EtOH twice. Keep coverslip under 96% EtOH. Tab2: Glycerol-DABCO mounting media (DABCO: 1,4-Diazabicyclo[2.2.2]octane) preparation: Use a 50 ml falcon tube: 2.5 ml of 0.2M TRIS-HCL, ph ml glycerol 0.5 g DABCO Wrap tube completely in foil to protect from light Mix on stirrer until dissolved, RT or 40 C (faster) Store at dark & 4 C. Tab3: Glycerol -NPG (n-propylgallate) Anti-fade Mounting Media preparation: Use a 50 ml falcon tube: 2.5 ml of 0.2M TRIS-HCL, ph ml glycerol 1.25 g n-propylgallate Wrap tube completely in foil to protect from light Mix on stirrer until dissolved, RT or 40 C (better/faster) Store at dark & 4 C. 4

5 Special requirements for each method: FRET / FLIM According to our experiences, we recommend Glycerol-DABCO mounting media (Tab2). If you optimized other mounting medium for FRET, no problem. As we know, Fluoromount G effectively kill the acceptor-photobleaching FRET measurement. DO NOT stain your specimen with DAPI or use any mounting media with DAPI when you want to do the CFP-YFP FRET measurement. If possible, please prepare three samples: unlabeled cells, cells labeled with the donor only, cells labeled with both the donor and the acceptor. SIM Staining/tagging No special requirements for expression vectors, immunostaining etc. Just select appropriate fluorophores according to system configuration: Ex: 405 / Em: ; Ex: 488 / Em: ; Ex: 561 / Em: ; Ex: 640 / Em: The DAPI staining we definitely recommend to do as a separate staining step followed with proper sample washing; do not use mounting media mixed with DAPI because of the background increasing. Mounting Because of intensive acquisition during 3D SIM, there is a higher requirement for fluorescence stability. The bleaching you can avoid by selection appropriate combination of staining and mounting. For immunostaining, we recommend to use Alexa Fluor (e.g. 488, 568 and 643) in combination with Glycerol-NPG mounting medium (Tab3). We do not recommend it for expression fluorescent proteins because of unwanted quenching. For expression vectors with fluorescent protein tag (e.g. GFP, mcherry etc.) we recommend Vecta Shield H1000 or Glycerol-DABCO (Tab1). For both expression and immunostaining you can also use Prolong Gold or Fluoromount-G, anyway the known disadvantage of hardening mounting media is loosing of the volume information caused by sample flattening. 5

6 If you are not sure or want to try other mounting media, you are very welcome to bring the sample stained but not mounted, just in Petri-dish under the PBS with 0.1% azide and mount them here to use several types of mounting media which we can provide you with. STED Staining/tagging Based on our system configuration (Leica TCS SP8 STED 3X with 660 nm depletion laser), the most suitable fluorophore is Alexa Fluor 555 for single staining. For double staining, possible combinations are AF488/AF555 (AF488 gives just poor superresolution), AF514/AF568 or AF514/AF546. Potentially working combination for triple staining is AF488/AF532/AF594. Our STED configuration allows to deal with expressed fluorescent proteins, but generally, the result depends on internal sample environment of particular sample. According to our direct experience, STED is doable with EYFP, dsred and partly with mcherry. Because the gated STED as a method is very sensitive and based on the fluorophore properties (fluorescence intensity, emission lifetime ) and the fluorophore properties depend on the specimen internal environment (ph, salts etc.), it is very recommended to bring several combinations. You are also very welcome to discuss the staining with us. You also can deliver samples stained just with primary antibody; we can provide you with suitable secondary antibody from our collection: goat AF488 (anti-rabbit, anti-mouse), 514 (anti-rabbit, antimouse), 532 (anti-mouse), 546 (anti-rabbit, anti-mouse), 555 (anti-rabbit, anti-mouse), 568 (anti-rabbit, anti-mouse) and 594 (anti-rabbit, anti-mouse). The DAPI staining we definitely recommend to do as a separate staining step followed with proper sample washing; do not use mounting media mixed with DAPI because of the background increasing. Mounting For Alexa Fluor we recommend Glycerol-NPG mounting medium (Tab3). For expression, we recommend Glycerol-DABCO (Tab1) or Vecta Shield H You can also use Prolong Gold for both immunostaining and expression, anyway the known disadvantage of hardening mounting media is the loosing volume information caused by sample flattening. FRAP Glass bottom Petri dishes are needed (3.5 mm, #1.5 thickness). If you are interested to try photoactivation method, keep in mind that the visualization of the structures is needed e.g. pagfp is visible after the activation, thus the region/structure of interest should be visualized by co-expression of some tag with different (dsred, mcherry etc.). 6

7 ATFM 2016 general partners Distributor of Thermo Scientific and Leica-microsystems instruments 7

8 Contact us: Course Coordinator: Ivan Novotny, PhD Light Microscopy Core Facility Institute of Molecular Genetics AS CR Videnska Praha 4 Czech Republic Phone: Fax: ivan.novotny@img.cas.cz Administration: Lenka Pišlová Microscopy Centre Institute of Molecular Genetics of the ASCR Videnska Prague 4 Czech Republic Phone: Fax: lenka.pislova@img.cas.cz 8