Exploring the Zeiss Axioimager A1 and Molecular Probe prefab cell and tissue section slides

1. PROJECT PREFAB FLUOR FRED MARTIN 11/06/2009 Dr. Giorgi BIOSC 2

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)

3. • 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)

4. SPECIMEN 1: FluoCells prepared slide #2: BPAE bovine pulmonary artery endothelial cells Image A - 20091105: Zeiss #1 20x NOTE: Considerable red channel bleaching exists all over this slide vs sample image

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

6. Image A - 20091018: Zeiss #1 20x SPECIMEN 2: FluoCells prepared slide #4: 16 μm cryostat section of mouse intestine

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

8. 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

9. BONUS SPECIMEN: FluoCells ® prepared slide #1: BPAEC, Zeiss 63x 20091016_INVG1_AREA3_CompositeAdj_jpg.jpg

10. BONUS SPECIMEN: FluoCells® prepared slide #1: BPAEC, Zeiss 40x 20091016_INVG1_AREA2_CompositeAdj.jpg 20091029_inv1_area4_CompositeRedMaxOK.jpg

11. BONUS SPECIMENS: FluoCells ® prepared slide #1: BPAE cells , Zeiss, 63x 20091016_INVG1_AREA2_CompositeAdj.jpg

13. 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)

14. Table 2. Spectral characteristics of the stains used in FluoCells® prepared microscope slides. FluoCells® prepared slide #1 (F36924) – BPAE cells Dye Ex* Em * DAPI 358 † 461 † Alexa Fluor® 488 phalloidin 505 512 MitoTracker® Red CMXRos 579 ‡ 599 ‡ FluoCells® prepared slide #2 (F14781) – BPAE cells DAPI 358 † 461 † BODIPY® FL goat anti–mouse IgG 505 513 Texas Red®-X phalloidin 591 608 FluoCells® prepared slide #3 (F24630) – mouse kidney section DAPI 358 † 461 † Alexa Fluor® 488 wheat germ agglut. 495 519 Alexa Fluor® 568 phalloidin 578 600 FluoCells® prepared slide #4 (F24631) – mouse intestine section Alexa Fluor® 350 wht grm agglutinin 346 442 SYTOX® Green stain 504 † 523 † Alexa Fluor® 568 phalloidin 578 600 FluoCells® prepared slide #6 (F36925) – muntjac skin fibroblasts Alexa Fluor® 488 phalloidin 495 519 Alexa Fluor® 555 goat anti–mouse IgG 555 571 TO-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.