PROJECT PREFAB FLUOR

FRED MARTIN11/06/2009 Dr. GiorgiBIOSC 2

FluoCells prepared slide #2 contains bovine pulmonary artery endothelial cells-BPAEC

>> Texas Red-X phalloidin is labeling the F-actin, >> BODIPY FL goat anti—mouse IgG antibody in conjunction with anti—bovine α-tubulin mouse monoclonal 236-10501 are labeling microtubules.>> DAPI is labeling the nucleus.

        

Texas Red®-X   Ex Peak: 595nm   Em Peak: 615nm

DAPI   Ex Peak: 358nm   Em Peak: 461nm

BODIPY® FL   Ex Peak: 505nm   Em Peak: 513nm

#2 Molecular Probes Sample Image (from mfg’s website)  

• FluoCells® prepared slide #4 (F24631) contains a 16 μm cryostat section of mouse intestine stained with a combination of:

>>Alexa Fluor® 350 wheat germ agglutinin, a blue-fluorescent lectin, was used to stain the mucus of goblet cells. >>Alexa Fluor® 568 phalloidin stained the filamentous actin prevalent in the brush border with red-fluorescent. >>SYTOX® Green nucleic acid stained the nuclei green.

Alexa Fluor® 350   Ex Peak: 347nm   Em Peak: 442nm

SYTOX® Green   Ex Peak: 504nm   Em Peak: 523nm

Alexa Fluor® 568   Ex Peak: 579nm   Em Peak: 604nm

#4 Molecular Probes Sample Image(from mfg’s website)

 

SPECIMEN 1: FluoCells prepared slide #2: BPAE bovine pulmonary artery endothelial cells

Image A - 20091105: Zeiss #1 20xNOTE: Considerable red channel bleaching exists all over this slide vs sample image

SPECIMEN 1: FluoCells prepared slide #2: BPAE – Images B –> E (All 20091105: Zeiss #1 20x)

Image A - 20091018: Zeiss #1 20x

SPECIMEN 2: FluoCells prepared slide #4: 16 μm cryostat section of mouse intestine

SPECIMEN 2: FluoCells slide #4: mouse intestine, Images BE (all 20091018: Zeiss1 20x, E@40x)

SPECIMEN 1: FluoCells prepared slide #2: BPAEC Overlay with DIC

20091105: Zeiss #1 20x RGB Merge + DIC NOTE: Considerable bleaching existed all over this slide

BONUS SPECIMEN: FluoCells® prepared slide #1: BPAEC, Zeiss 63x

20091016_INVG1_AREA3_CompositeAdj_jpg.jpg

BONUS SPECIMEN: FluoCells® prepared slide #1: BPAEC, Zeiss 40x

20091016_INVG1_AREA2_CompositeAdj.jpg

20091029_inv1_area4_CompositeRedMaxOK.jpg

BONUS SPECIMENS: FluoCells ® prepared slide #1: BPAE cells , Zeiss, 63x

20091016_INVG1_AREA2_CompositeAdj.jpg

Reference Information:

1) All post acquisition processing done on ImageJ2) All data for Molecular Probes sllides taken from Invitrogen websitehttp://www.invitrogen.com/site/us/en/home/Global/invitrogen-search-results.html?searchTerm=FluoCells+prepared+slide+%

3) All spectra data and graphs taken from Omega Filters webtool:https://www.omegafilters.com/curvo2/index.php

4) Things I learned in this project:

a. Know/learn the “expected look” of the specimen before starting imaging: without a baseline, it not possible to determine color range, colocalization tones, and suspect bleached areas.b. Always refocus on the monitor image, and refocus each channel.c. It’s possible to take good images quickly…and easier to take bad ones quickly.d. Acquiring images with camera in BW mode works for me.e. Be sure you have reset/understand everything on your baseline system (hardware + software) before you start imaging .f. The best images come from good composition, good focus, and good exposure: Pre-acquisition is key!g. You can’t really fix problems in step (f) post acquisition.

FluoCells® prepared slide #1 (F36924) contains bovine pulmonary artery endothelial (BPAE) cells stained with a combination of fluorescent dyes.

>> Mitochondria were labeled with red-fluorescent MitoTracker® Red CMXRos

>> F-actin was stained using green-fluorescent Alexa Fluor® 488 phalloidin

>> Blue-fluorescent DAPI was used to label the nuclei.

Additional reference Info: Slide #1

#2 Molecular Probes Sample Image (from mfg’s website)  

Table 2. Spectral characteristics of the stains used in FluoCells® prepared microscope slides.FluoCells® prepared slide #1 (F36924) – BPAE cellsDye Ex* Em *DAPI 358 † 461 †Alexa Fluor® 488 phalloidin 505 512MitoTracker® Red CMXRos 579 ‡ 599 ‡

FluoCells® prepared slide #2 (F14781) – BPAE cellsDAPI 358 † 461 †BODIPY® FL goat anti–mouse IgG 505 513Texas Red®-X phalloidin 591 608

FluoCells® prepared slide #3 (F24630) – mouse kidney sectionDAPI 358 † 461 †Alexa Fluor® 488 wheat germ agglut. 495 519Alexa Fluor® 568 phalloidin 578 600

FluoCells® prepared slide #4 (F24631) – mouse intestine sectionAlexa Fluor® 350 wht grm agglutinin 346 442SYTOX® Green stain 504 † 523 †Alexa Fluor® 568 phalloidin 578 600FluoCells® prepared slide #6 (F36925) – muntjac skin fibroblastsAlexa Fluor® 488 phalloidin 495 519Alexa Fluor® 555 goat anti–mouse IgG 555 571TO-PRO®-3 stain 642 † 661 †

* Fluorescence excitation (Ex) and emission (Em) maxima, in nm. † When bound to DNA. ‡ Determined in methanol; values may vary somewhat in cellular environments.