2. Introduction
Culture, dilution and staining of Chinese Hamster Ovary
(CHO) cells
Zebrafish embryo staining
Confocal microscopy and image processing(using Image J)
of the above
3. Flow Chart: Culture & Staining of CHO cells
Cells
DMSO (3)
Nocodazole (2)
Fix 3.7% HCHO,15min20min,37°C
0.1% Triton X-100,5 min2 X PBSAllexa 568 Phalloidon
(1:100), 30 mins
3 X PBS
Blocking
Sol,30 min
1° Ab (α tubulin
1:500), 30 min
3 X PBS
2° Ab (Alexa
488,1:100), n=4
No 2° Ab, n=1,
DMSO
30min
3 X PBSStore in
dark
4. CHO Cells – Confocal Microscopy
1. Raw Image 2. Red Channel (F Actin) 3. Green Channel (Tubulin)
4. Z Project 5. Merged & Processed 6. Control
5. Processed, Magnified Image in Merged Channels –
Normal CHO cell
Green Stains ~
Tubulin
Red Stains ~
F-Actin
6. Nocodazole treated CHO cells - Confocal Microscopy
3. Green Channel (Tubulin)2. Red Channel (F Actin)1. Raw Image
4. Z Project 5. Merged & Processed 6. Control
7. Nocodazole treated CHO cells –
Merged, Magnified & Processed Image
Red Staining ~ F-
Actin
Green Staining ~
Tubulin
Nocodazole typically
depolymerizes microtubules;
here, we can see tubular
structure is destroyed (green
staining) to a more
homogenous, scattered tubulin
dimers spread out in the cell
cytoplasm
8. Quantification of F – Actin in normal
CHO and Nocodazole treated cells
Normal CHO ~ F-Actin Nocodazole treated CHO ~ F Actin
9. Quantification of tubulin in normal CHO and
Nocodazole treated cells
Normal CHO ~ Tubulin Nocodazole treated CHO ~ tubulin
11. Quantification
Formula: Weighted Mean Intensity of 4 cells (grayscale/pixels) – Background
Correction
F-Actin: Normal Cell > Mean Intensity = 56.29
Nocodazole treated cell > Mean Intensity = 20.189
% Decrease in area > 64% after drug treatment
Tubulin: Normal Cell > Mean Intensity = 84.30
Nocodazole treated cell > Mean Intensity = 59.40
% Decrease in area > 29.53 % after drug treatment
Conclusion: Nocodazole causes depolymerization in microtubules; hence, we
noticed a 29.53 % decrease in area in tubulin content after drug treatment.
Interestingly, percentage decrease of 64% was more significant in F-Actin then
tubulin, proving that nocodazole not only affects microtubules, but also attacks F-
Actin. This phenomenon also proves that tubulin and F-Actin are not too
biologically different and made of moieties where effects of nocodazole can be
easily observed.
12. Processing of Given Image Stacks – Exercise in
Image J
Raw Image Processed Image
13. Model Image of ZebraFish Embryo (3 day old)
Source:www.nimr.mrc.ac.uk/ devbiol/xu/zebra_image/
14. Anatomy of a 72 hr old embryo (dorsal view)
Source: zfin.org/zf_info/ anatomy.html
Hair cells (tether
cells) bind seeding
particles, thereby
localizing otolith
formation. Tether
cells usually occur
in pairs at the
anterior and
posterior ends of the
ear. tether cells
initially appear
immature and do
not acquire the
characteristics of
mature hair cells
until approximately
21.5 h.
15. Zebrafish Embryo Staining Methods -
Flowchart
Day 1: Gently remove Chorions From Embryo’s
Anthesthetize Embryos (1/20 volume 20X Tricaine)
Transfer To 20 ml Scintillation Vial
Repeat steps 1 and 2 for all time points
Move embryos to moving shaker
PBS 0.1% Triton X-100 (2X 30min)
Transfer embryos to 24 well dish
PBT rinse (PBS + 1% Triton, 1hr on shaker)
16. Zebrafish Embryo Staining Methods –
Flowchart (continued)
PBT Rinse (PBS + 0.1% Triton X-100 2X 30 min)
Blocking Solution (1 hr on shaker)
Primary Antibody incubation (1:100 tubulin, 1:250 HCS)
Incubate overnight (dark, in fridge)
Day 2: PBT rinse (PBS + 0.1% Triton X-100 3X 30 min) ~ done by TA
Secondary Antibody Incubation (Alexa 488 for tubulin 1:500,
Alexa 568 for HCS-1 1:500
Store in dark at 4 degrees C, overnight
17. Zebrafish Embryo Staining Methods –
Flowchart (continued)
Day 3: PBT rinse (PBS + 0.1% Triton X-100 3X 30min)
PBS rinse (1X 30 min)
70% glycerol in PBS on shaker (30 min)
Remove egg yolk with sharp tweezers,
under microscope
Place drop VectaShield Mounting Medium
Pick up single embryo, place in slides and
place cover slips
Stpre Coverslip in fridge, away from light
20. Zebrafish Embryo – Processed & Merged Images (Z
project)
Control
Tether Cells
Lateral Line System
21. Conclusion
Cells and embryos are successfully stained and
photographed under confocal microscope (using 2D &
3D imagery)
Positive results were obtained when compared with
controls
Images were effectively processed using Image J software