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One-Hundred Imaging TipsAs seen on the "Cell Imaging" Facebook Page

Jason A. KilgoreMolecular Probes Tech Support, Life Technologies

100 Imaging Tip of the Day: If your tissue is already labeled with afluorescent dye (such as neuronal tracers or injected cells tracked with dyes),be aware that paraffin processing may extract the dye. Consider cryosectionsinstead.

99 Imaging Tip of the Day: Use of BrdU is a well-established method fordetermining cells that have proliferated, but it requires denaturing DNA withHCl, which can affect cell morphology, and the use of antibodies for detection,

which requires time and can add background. Consider Click-iT EdU, whichincorporates the same way but doesn't need HCl or antibodies, just a 20-minute"click" reaction to detect: www.invitrogen.com/edu 

98 Imaging Tip of the Day: Tissue is particularly bad for autofluorescence, butparaffin sections are worse than cryosections. Consider using autofluorescencereduction methods (like sodium borohydride treatment), amplification methods(such as avidin-biotin), and avoiding direct conjugates (which are dimmer).

97 Imaging Tip of the Day: Is your dye stock in DMSO? Be aware that DMSOwill freeze in the refrigerator and condense a bit, giving the impression that

you received a solid or too low a volume. DMSO takes a while to thawcompletely, so give it time at room temperature or with gentle warming beforeopening and using. 

96 Imaging Tip of the Day: Autofluorescence is a particular problem for plantsamples, due to phycobiliproteins, chlorophyll, xanthophylls, carotene, andvarious flavinoids fluoresce in a variety of wavelengths. Make sure to have a no-dye control to test for this.

Images of plant autofluorescence:http://www.olympusconfocal.com/gallery/plants/index.html 

95 Imaging Tip of the Day: When imaging cells with microplates, choose atype with the least autofluorescence in your wavelength by testing them firstwith a water blank. Also, opaque walls will prevent bleedthrough betweenwells.

94 Imaging Tip of the Day: DNase I conjugates have been used for labeling G-actin in fixed and permeabilized cells, and has relatively low affinity to F-actin

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(unlike phalloidin conjugates). However, DNase I conjugates may label nuclei,too.

93 Imaging Tip of the Day: DiOC6(3) is a traditional dye for labelingendoplasmic reticulum, but it's not very selective, labeling mitochondria and

other structures, too. ER-Tracker dyes are far more selective.

92 Imaging Tip of the Day: Good link for info on using antibiotics andantimycotics in cell culture: http://bit.ly/iKyKnv 

91 Imaging Tip of the Day: Need a quick sample to throw on the fluorescentscope to demonstrate the hardware? Commercial slides are available, likeFluoCells cell and tissue slides or FocalCheck bead slides. You can also makequick samples using autofluorescent pollen or flower petals.

90 Imaging Tip of the Day: Calcein AM is often used as a live cell indicator or

for short-term cell tracking and cell mobility testing. It is retained for just afew hours and isn't bound covalently (and thus isn't as likely to perturb cells),but it can be pumped out of cells over time, leading to decreased signal.

89 Imaging Tip of the Day: A link for general troubleshooting of whole mountimmunolabeling problems:http://www.abcam.com/ps/pdf/protocols/whole_mount_troubleshooting.pdf  

88 Imaging Tip of the Day: Useful numbers for cell culture (seeding densities,volumes, confluencies, etc): http://bit.ly/mjOnnr 

87 Imaging Tip of the Day: When a protein is over-labeled, the proximity of the dyes to each other can lead to quenching of the overall fluorescence,called "dye-dye quenching." If this is a problem reduce your dye-to-proteinmolar ratio. Some assays purposely self-quench proteins to show a de-quenching fluorescence upon completion of the assay (such as this one:http://probes.invitrogen.com/media/pis/mp12052.pdf  ).

86 Imaging Tip of the Day: AM ester dyes and indicators can sometimes havedifficulty in getting a uniformly-dispersed final solution or uniform loading. If this happens, use of Pluronic F-127 (a non-ionic surfactant polyol) can help.PowerLoad, too, which is an optimized formulation of Pluronic compounds.

85 Imaging Tip of the Day: Be aware that when you conjugate a protein witha dye, you always risk losing some or all of the protein's functionality by sterichindrance of active sites (especially with high degree-of-labeling) or changes inprotein conformation (if reduction is required, for instance).

84 Imaging Tip of the Day: Some traditional flow cytometry dyes are not asuseful for microscopy, usually due to photostability problems or filter

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mismatch. Examples include R-phycoerythrin (PE), Pacific Blue, andallophycocyanin (APC).

83 Imaging Tip of the Day: If you are labeling with an indicator dye, such asH2DCFDA, and your initial signal looks good, but after a certain time period

looks dimmer, it may be that the dye is being actively extruded out of thecells. Use of probenecid may help stop the active extrusion and retain the dyein the cytoplasm.

82 Imaging Tip of the Day: If you are using a avidin-biotin amplificationstrategy, be aware that cells have endogenous biotin which will need to beblocked (particularly in mitochondria). Commercial endogenous biotin blockingkits are available.

81 Imaging Tip of the Day: Qdots are the most photostable fluorophores. Butfor archiving of slides, you need to mount in Qmount mounting medium. All

other mountants will lead to loss of initial intensity over days to weeks. 

80 Imaging Tip of the Day: A protocol for embedding tissues forcryosectioning:http://molecularprobestechnologynetwork.community.invitrogen.com/docs/DOC-1042 

79 Imaging Tip of the Day: Causes of aggregation for microspheres:http://molecularprobestechnologynetwork.community.invitrogen.com/docs/DOC-1041 

78 Imaging Tip of the Day: How to choose the right filter set:http://bit.ly/iHcBCW 

77 Imaging Tip of the Day: An example protocol for immunocytochemicallabeling of cultured cells:http://molecularprobestechnologynetwork.community.invitrogen.com/docs/DOC-1031 

76 Imaging Tip of the Day: Causes and fixes of photobleaching summary formicroscopy:http://molecularprobestechnologynetwork.community.invitrogen.com/docs/D

OC-1043

75 Imaging Tip of the Day: Good technical note about the structure andclassification of antibodies: http://bit.ly/hZHwLm 

74 Imaging Tip of the Day: Good article from BioProbes 62 on autophagyassays for live cells: http://bit.ly/iTRnqH 

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73 Imaging Tip of the Day: If you are imaging with a plate reader, make sureto use a brand of plate that has low autofluorescence in your wavelength, toimprove signal-to-background ratio. If in doubt, scan a water blank for eachbrand that you have available and use the one with the lowest value. Opaquewalls are a plus, to prevent cross-talk between wells.

72 Imaging Tip of the Day: Phalloidin conjugates are an excellent way to labelF-actin in fixed and permeabilized cells; no antibody needed. Don't fix withsolvents, though.

71 Imaging Tip of the Day: Great article summarizing fluorescent dyes for usein plant samples: http://bit.ly/m2nP7m 

70 Imaging Tip of the Day: Good table of scavengers of reactive oxygenspecies: http://bit.ly/kh8lCH 

69 Imaging Tip of the Day: Problem with your antibody conjugate coming off its target (off-rate) after labeling and mounting your sample? Consider post-label fixation with formaldehyde to cross-link it in place, mounting in asolidifying mounting medium, and storing cold.

68 Imaging Tip of the Day: Examine your fluorescent filters regularly. If thecoating is irregular, splotchy, or has a "bull's-eye" pattern, then it is degrading.This will lead to a wider bandwidth, which will lead to more bleedthroughproblems between imaging channels. 

67 Imaging Tip of the Day: For AM ester and diacetate dyes, remember that

different cell types have different levels of esterase activity, so you'll need tooptimize label time and dye concentration for your system.

66 Imaging Tip of the Day: There is no reliably *fixable* organic dye forlabeling lysosomes. However, CellLight Lysosome fluorescent protein productswill transiently transfect mammalian cells for lysosome labeling using aBacMam construct and can be subsequently formaldehyde-fixed (and is non-perturbing to viability). 

65 Imaging Tip of the Day: Good site for introduction to basic statistics:http://www.usablestats.com/ 

64 Imaging Tip of the Day: If you are using lipophilic dyes, subsequentexposure to detergents or solvents will delipidize the sample, leading to loss of the dye, including methanol in methanol-stabilized formaldehyde (formalin) orpermeabilization with Triton X-100.

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63 Imaging Tip of the Day: If a mounting medium is advertised as "antifade",check to see why. If it's just because it hardens, that will help, but likely won'tbe as effective as having an antioxidant present as well. 

62 Imaging Tip of the Day: Important criteria for choosing a calcium indicator

include desired Kd value (calcium concentration range), desiredexcitation/emission wavelength, and whether you want ratiometric or non-ratiometric. Here is a good table for many dyes: http://bit.ly/gFldvq 

61 Imaging Tip of the Day: An interesting guide to using calcium indicators:http://bacterio.cbm.uam.es/confocal/manuales/calcium_imaging_Eamnet_SCastel.pdf  

60 Imaging Tip of the Day: When labeling cells with a succinimidyl ester (suchas CFDA SE), be aware that the reagent will bind to any amine groups,including on the cell surface or a poly-L-lysine coating on the substrate, or

proteins in media.

59 Imaging Tip of the Day: A good review article for neuronal tracing:http://www.neuroanatomy.org/2003/002_005.pdf . Another for how to do theanalysis:http://www.imagescience.org/meijering/publications/download/cyto2010.pdf 

58 Imaging Tip of the Day: Excellent site for all things related to microscopes:www.microscopyu.com.

57 Imaging Tip of the Day: The most nuclear-selective dyes for cells are DAPI

and Hoechst dyes. All others will label both DNA and RNA, though theirselectivity for one or the other will vary between dye options and conditions.For those others, an RNase wash first (for fixed and permeabilized cells) willtypically be needed to insure clean nuclear labeling.

56 Imaging Tip of the Day: Problems with your cell culture? Here is atroubleshooting guide:http://tools.invitrogen.com/content/sfs/appendix/Cell_Culture/Cell%20Culture%20Troublshooting%20Guide.pdf  

55 Imaging Tip of the Day: Don't underestimate the importance of good

ergonomics in the lab to prevent injuries, such as low-impact pipetters,comfortable lab stools, and moving things on your lab bench to be within easyreach. Take frequent stretch breaks, and adjust your microscope eyepiecesand chair height appropriately. Here is a good link for other suggestions:http://dohs.ors.od.nih.gov/labs.htm 

54 Imaging Tip of the Day: Trypsin is typically used to detach adherent cells.But this temporarily disrupts the plasma membrane, which will cause "false

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dead" readings with dead cell indicators, leakage of esterases for AM esterdyes, or false-positive annexin V readings. Wash out trypsin well and allow aresting period before labeling.

53 Imaging Tip of the Day: Qdot quantum dots can be quenched by heavy

metals, casein-containing blocking solutions, or most hydrophobic barrier pens(the ImmEdge pen sold by Vector Labs is one that doesn't quench).  

52 Imaging Tip of the Day: DAPI is a great nucleic acid stain, but it is onlysemi-permeable to live cells; some cell types work fine, others not. Considerusing Hoechst 33342, which labels DNA the same way and has the samespectrum, but is far more cell-permeant.

51 Imaging Tip of the Day: There are a lot of apoptosis assays, mainly formicroscopy of live cells, HCS, or flow cytometry. See page 16 in BioProbes 52for a list of some options for various stages of apoptosis:

http://www.invitrogen.com/site/us/en/home/support/Newsletters-and-Journals/BioProbes/BioProbes-Archive/Bioprobes-52.html .

50 Imaging Tip of the Day: For taking digital microscopic images, an optimizedexposure time is typically one which allows the brightest pixels of the area of interest without over-saturating the pixel values, for a given filter andobjective.

49 Imaging Tip of the Day: The lower you excite Qdot nanocrystals the better,regardless of emission wavelength. Use your lowest available laser line orexcitation filter. UV or 405nm is optimal. If you don't have UV or 405, 457nm

laser works great, or even 488nm, for all Qdots.

48 Imaging Tip of the Day: Nucleic acid stains, like DAPI or propidium iodide,do not covalently bind to DNA, and they have an off-rate over time. Thus, theyare not necessarily well-retained after fixation, are not "fixable," and shouldn'tbe used as long-term cell tracers. 

47 Imaging Tip of the Day: Be sure to rest and get enough sleep. Lack of sleepreduces your powers of observation. It can also lead to clumsiness, and wescientists often work with dangerous biohazards, chemicals and solvents.(Sleeping with your head against the microscope in that dark imaging room

doesn't count!).

46 Imaging Tip of the Day: Doing two-photon microscopy? Here is a good linkfor two-photon absorption cross-sections for dyes:http://www.drbio.cornell.edu/cross_sections.html

45 Imaging Tip of the Day: Antigen retrieval techniques, to unmask antigens insamples for immunolabeling, typically involve heating the sample, but the best

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instrument, buffer, and timing differ from antigen to antigen. Here is oneguide to different techniques:http://www.ihcworld.com/epitope_retrieval.htm. Not all antigens require it.

44 Imaging Tip of the Day: Interesting interactive page illustrating cell

structure and organelles:http://learn.genetics.utah.edu/content/begin/cells/insideacell/ 

43 Imaging Tip of the Day: Lipophilic cyanine dyes, like DiI C18, have oftenbeen used for tracking cells. Be aware, though, that these dyes float inmembranes and can be transferred from one membrane to another if cells fusein any fashion, and can be lost from the cell if the membranes arecompromised or permeabilized. 

42 Imaging Tip of the Day: Make sure to clean your objective very well if youchange from one immersion oil to another. If two unlike oils mix, not only can

there be refractive index problems, but some will react with each other toform a sticky substance that can damage your lens.

41 Imaging Tip of the Day: Is your fluorescent dye solution a slightly differentcolor than older lots, when viewed by eye in room light? Don't worry, the colorcan vary in production from lot-to-lot, and isn't important for most fluorescentdyes. What matters is the fluorescent properties and functionality.

40 Imaging Tip of the Day: Need to make your own conjugate using a reactivedye (as opposed to buying a kit)? Here is a good selection tool:http://igene.invitrogen.com/products/selector/dyes 

39 Imaging Tip of the Day: Lectin conjugates will label organelles in fixed andpermeabilized cells, such as ER and Golgi bodies, but be aware that theirselectivity varies between cell types. Often there are better dye options.

38 Imaging Tip of the Day: Autofluorescence of samples can interfere withfluorescent dyes. Make sure to have an unlabeled sample for anautofluorescence control, imaged the same way. Here's a good link for ways toreduce autofluorescence:http://www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.pdf .

37 Imaging Tip of the Day: A good site discussing cleaning of microscopylenses: http://www.microscopy-uk.org.uk/mag/artfeb04/cdclean.html 

36 Imaging Tip of the Day: Regarding latex lab glove allergy, here is a goodlink on the topic:http://www.kcdigestivehealth.com/docs/glovemetender.html . Nitrile is agood option. Though rare, be aware that some people can have an allergy tonitrile gloves, too.

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 35 Imaging Tip of the Day: Beware of very new or very old mercury bulbs:their intensities can waver, leading to quantitation errors. Let new bulbs burnin before using, and replace old bulbs according to manufacturer guidelines formax number of hours (old bulbs will grow dim as well).

34 Imaging Tip of the Day: If you want to visualize an entire GFP-transfectedembryo or whole-mount with a general counterstain, most lipophilic cyaninedyes will work, but a great one is BODIPY TR methyl ester, which won't overlapspectrally and can label live or formaldehyde-fixed samples.

33 Imaging Tip of the Day: When calling for Tech Support for a specificproduct, be sure to have a catalog number handy. If it is for troubleshooting aproduct you already possess, be sure to have the lot number and order datehandy, too.

32 Imaging Tip of the Day: Want to quench dyes in the extracellular media,but not in the cells? Trypan blue is a common option, for instance forphagocytosis assays.

31 Imaging Tip of the Day: Some immersion oils can have autofluorescentbackground. If you do fluorescent microscopy, make sure your immersion oil isvalidated or designed for fluorescence, and it is a good idea to comparebackgrounds of different oils initially on your system.

30 Imaging Tip of the Day: For finding dyes to match your flow cytometry laserlines, here is a really good guide, which lists most of the common flow

cytometers and matches dye options:http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/probes.Par.83037.File.dat/O-072942-Flow-Instrumentation-Guide.pdf  

29 Imaging Tip of the Day: Always use gloves in the lab, even with buffers, andhave a variety of choices. But which gloves? Make sure the type of glove youuse is compatible with your chemical or solvent. Here is a link to glovecompatibility charts, by company:http://www.ehs.ufl.edu/Lab/CHP/gloves.htm 

28 Imaging Tip of the Day: Safety first! Anytime a new person starts work in

your lab, the first thing you should do is explain the safety rules and familiarizethem with locations for safety equipment (emergency phone number list,evacuation route, eyewash, emergency shower, fire extinguisher, first aid kit,spill kit, gloves, biohazard and chemical disposal stations).

27 Imaging Tip of the Day: A great science calculator for your phone or iPodTouch, called DailyCalcs, for molarity calculations, dilutions, formula weight,cell culture specs, and unit conversions:

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http://itunes.apple.com/us/app/dailycalcs-science-calculator/id353223512?mt=8 

26 Imaging Tip of the Day: An amazing resource for spectral information onjust about any fluorescent dye: PubSpectra:

http://home.earthlink.net/~pubspectra/ 

25 Imaging Tip of the Day: Good sites for finding primary antibodies:Linscott's Directory: http://tinyurl.com/4oaq5dq Biocompare: http://tinyurl.com/4jwbx64 Invitrogen Primary Ab Selection Tool: http://tinyurl.com/4mbj9ya 

24 Imaging Tip of the Day: When choosing a FRET pair, consider both thespectral overlap of the donor and acceptor dye, as well as the R(0) value [thedistance in Angstroms at which fluorescence resonance energy transfer fromthe donor dye to the acceptor dye is 50% efficient (Förster radius)]. Here is a

table of R(0) values for some Alexa Fluor dye pairs, for instance:http://bit.ly/f1u6Yo  Technical note on FRET and other R(0) values: http://bit.ly/fzqNkc 

23 Imaging Tip of the Day: Photobleaching of samples using confocal? If useof an antifade mountant or a more stable dye isn't an option, try adjustingsettings. A faster scan rate and less average dwell time can help, but thetradeoff is a loss of resolution. You can also lower the laser voltage, thoughinitial intensity will be reduced. 

22 Imaging Tip of the Day: If you image samples in buffer or a non-curing

mounting medium, you have to seal the coverslip edges. We recommend usingmelted paraffin, (applied with a cotton-tipped applicator stick) which hardensinstantly and is hydrophobic. Many labs use clear nail polish, but this hassolvents which can kill live cells and quench some dyes, like GFP.

21 Imaging Tip of the Day: Tracking bacteria with dyes can be accomplishedby using CFDA SE, which will label mainly intracellular proteins in live bacteria,or by using a reactive dye such as an Alexa Fluor succinimidyl ester derivative,which will label proteins on the surface of the bacteria (alive or dead).

20 Imaging Tip of the Day: FM dyes (like FM 1-43) are lipophilic stains good for

endocytosis or synaptic vesicle studies. Some have been used for plasmamembrane labeling of cultured cells, but this can be problematic due toextremely fast endocytosis and poor retention after washing. Better options:CellMask Plasma Membrane Stains, wheat germ agglutinin conjugates, orCellLight BacMam products. 

19 Imaging Tip of the Day: If you are testing a drug treatment with afluorescent assay, make sure to have a no-dye control with and without the

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drug, as some drugs can have fluorescence of their own that can bias yourreading. 

18 Imaging Tip of the Day: Bubbles in your mounting medium uponcoverslipping? Could be from the angle you mounted it, air trapped in your

sample (particularly tissue), or bubbles in the mountant stock solution.Combat by mounting the coverslip at an angle, pulling a slight vacuum on yourtissue in buffer prior to mounting, and spinning the mountant stock prior tousage. A few mounting media brands can also outgas upon curing. 

17 Imaging Tip of the Day: Don’t do your live cell fluorescent imaging inmedia that contains phenol red, as it can quench many dyes. Most commonmedia are available without it, or you can image in HBSS or DPBS.

16 Imaging Tip of the Day: For testing cell fusion assays, it is important tomark the two cell populations with tracking dyes. Perhaps the best option is to

use lipophilic cyanine dyes, like DiO C18 (green) for one population and DiI C18(red-orange) for the other population, labeling membranes. If successfullyfused, the fused cells will appear in both colors with flow cytometry (or yellow,by eye).

15 Imaging Tip of the Day: A typical imaging lab can produce thousands of images a year. To avoid confusion, here is an example template we've used forlabeling images: "12_AbA5ug_FITC_60x_021611a.tif" would refer to an image of slide 12, testing "antibody A" at 5 ug/mL, imagined in the FITC channel at 60x,on February 16, 2011, and field "a" of the sample (if you image multiple fieldsper sample, i.e. a-c). 

14 Imaging Tip of the Day: Are you using a curing, aqueous mountant butlater realize you need to remove the coverslip, maybe to re-stain the sample orget rid of bubbles? Simply soak the slide in warm PBS for 30-120 minutes withgentle rotation. The mountant will dissolve and the coverslip will slide off.Wash well and proceed with restaining and remounting.

13 Imaging Tip of the Day: Microspheres can sometimes aggregate in the vial,but it's usually reversible. Potential causes: freezing, microbial contamination,high salt concentration, extremes in pH, high DMSO concentration (>2%), orsimply small diameter. Combat with appropriate storage, bath sonication, or

small concentration of detergent (such as 0.1% Tween-20).

12 Imaging Tip of the Day: Don't freeze your Qdot nanoparticle products;they'll aggregate irreversibly. This can happen during shipment during snowyweather too. Spin them down before using. If there's a pellet, they're probablyaggregated. 

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11 Imaging Tip of the Day: If you're looking for a tool to help choose dyes forspecific organelles, try the Stain Your Own Cell online tool, where you can mixand match color and dye options for live and fixed cell labeling and see them ina simulated cell: http://www.invitrogen.com/cellpainting

10 Imaging Tip of the Day: If you wish to label cell membranes in live orformaldehyde-fixed (but unpermeabilized) cells, be sure to specify plasmamembrane or all membranes. Lipophilic cyanine dyes, such as DiI C18, willlabel all membranes of the cell, while wheat germ agglutinin conjugates,concanavalin A conjugates, or CellMask™ plasma membrane stains are moreselective for the plasma membrane.

9 Imaging Tip of the Day: Propidium iodide (non-cell permeant) and Hoechst33342 (live cell permeable) nucleic acid stains work great for nuclear labelingof mammalian cells, but in plant cells they will also label the cell wall.

8 Imaging Tip of the Day: When choosing a dye, make sure it matches yourfilter specs or laser line. There are handy tools online. One is theSpectraViewer tool, where you can display the spectra for up to five dyes at atime and put in your filter or laser wavelengths to check for overlap:http://www.invitrogen.com/spectraviewer 

7 Imaging Tip of the Day: Problems with apparent secondary antibodybackground? Be sure to run a secondary-only control to insure the problem isn'tfrom the primary, try increased protein blocking (such as 6% BSA/10% normalserum), or reducing the secondary concentration or time, and consider thatsome background can be due to charge-based binding (which can be blocked

with Image-iT® FX signal enhancer).

6 Imaging Tip of the Day: Have two primary antibodies of the same species forthe same sample? To avoid cross-label, you can use isotype-specificsecondaries if they are of different isotypes (IgG1, IgG2a, IgG2b), or you canmake direct conjugates. Here is help in choosing a kit for making directconjugates: http://www.invitrogen.com/ablabeling

5 Imaging Tip of the Day: When choosing a primary antibody, be sure to checkwhich techniques they are validated for. It is typically noted in the productmanual or certificate of analysis. For instance, antibodies validated for

Western blotting or ELISA are not always successful for immunocytochemistryor immunohistochemistry techniques.

4 Imaging Tip of the Day: When considering the range of concentration to testfor your primary or secondary antibody, be aware that instrument sensitivity isalso a concern. For instance, microscopy is typically less sensitive than flowcytometry and often requires a higher concentration.

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3 Imaging Tip of the Day: If you are labeling live cells with diacetate dyes,such as many ion indicators or cell tracking dyes, which are cleaved bycytoplasmic esterases to become active, make sure you do not label in thepresence of serum, which has esterase activity and will prevent uptake of thedye.

2 Imaging Tip of the Day: Having a uniform intensity across your field of viewis important, particularly for quantitation. To correct for this, you can make auniform intensity standard slide by making a solution of a reference dye inbuffer and putting it under a coverslip, or purchase ready-made plasticuniformity slides from distributors.

1 Imaging Tip of the Day: Photobleaching problems? For fixed samples, usingan antifade mounting medium is recommended (such as ProLong Gold®). Forlive samples, try using a second, more stable dye (such as Hoechst 33342) tofind and orient the cell first, before switching over to your problematic dye to

snap a picture, thus limiting the time it is being exposed and fading.