Imaging

Blood Vessel Dihydroethidium Dhe Quantification Using Image J

A detailed protocol for DHE signal quantification on blood vessel samples using Image J.

I use Leica TCS SP5 Confocal Microscope for imaging, red channel for DHE signaling (pay attention to the special excitation and emission parameters) and green channel for collagen auto-fluorescence (I used the default setting for Alexa 488). Here is an example of DHE staining on mouse aorta.

 

 

Procedure

Image 1

Image 2

Image 3
 

Image 4

  1. Open image in Image J.
  2. Go to Image→Color→Split Channel. You will get three images in gray scale showing signals in red (image 1), green (image 2) or blue channel. I don’t have anything in blue channel, so just ignore the split result in blue channel. As you will see, I do have auto-fluorescent signal in the red channel, which was not showing obviously in the overlay. This is why I do not recommend only capture DHE signal and use the “measure” function, which will count both DHE signal and auto-fluorescent signal. (There is an exception. If you have super strong signal, you could use very low exposure time to avoid capturing the auto-fluorescent signal.)
  3. Go to Process→Image Calculator. Subtract the image green (image 2) from image red (image 1).
  4. If the intensities of auto florescence are similar in these two channels, you will get rid off the auto fluorescence in red channel in the result (a new image will pop up, image 3). If you still have auto fluoresce in the result, just simply do the subtraction again. Make sure you do everything the same for images in all groups. 
  5.  Now we are ready to count the intensively of DHE signal. Click the result image (image 3), Go to Analyze → Measure, you will get integrated intensity for DHE signal.
  6. You may notice, when you take images, the amount of blood vessel in each image may be different. For normalization, I count the area of auto fluorescence to represent the sample size in the image using image 2. Click image 2, go to Process→ Binary→ Make Binary (image 4). The binary step takes out the intensity variation of auto fluorescence in each image. Now, click image 4, Go to Analyze → Measure. Use the integrated intensity (represents area of auto fluorescence) for normalization.
  7. Calculate DHE signal / auto fluorescence signal ratio and compare each group.

Automatic Counting Of Fluorescently Stained Cells Using Microscope And Photoshop Or Imagej

This protocol describes semi-automated cell counts using fluorescently labeled cells, a hemocytometer and ImageJ software.  The hemocytometer is not needed if you have already calibrated the areas of images taken with your microscope and camera.  Our setup automatically imports images in iPhoto and loads them into Photoshop with a double-click.  However, you could also import and crop the images directly into ImageJ if you prefer.

Materials
Cells in media
EtOH
1x P.I. (100 µg propidium iodide / mL PBS)
Hemocytometer
U.V. fluorescent microscope with camera
Computer with ImageJ and image cropping software.

Procedure
Fix Cells by diluting them 50% in EtOH.
1.  To an eppendorf add 0.1 mL cells in PBS or media
2.  Add 0.1 mL of 100% EtOH while vortexing.
3.  Spin at 3,000 RPM x1 min.
4.  Aspirate off supernatant and respend cells in 0.1 mL of 1x P.I.  Mix, incubate 2 min. 

Photograph cells on u.v. microscope
5.  Add 10 µL of stained cells to Hemocytometer.
6.  Place on U.V. microscope. (If the cells are too crowded then you may need to further dilute them in P.I. or PBS)
7.  Photograph 1 large square of hemocytometer with brightfield.
8.  Without moving the stage or changing the zoom, photograph the same area under u.v. light with red filter.  

Import and crop photo (e.g. using iPhoto and Photoshop)
9.  Import photos from the camera to the computer (e.g. with iPhoto).  Double click an image to open image in Photoshop.
10. In Photoshop, use the marquee tool measure the size of large square of the brightfield hemocytometer image.

(A large square on the hemocytometer is 1mm x 1mm x 0.1mm = 0.1 µL)

11. Crop the u.v. fluorescent image of cells to same size as a large square.
12. Convert to grayscale (Image > Mode > Grayscale).
13. Save file as .jpg image (medium resolution).

Count cells in cropped image using ImageJ
14. Open .jpg file with ImageJ software.
15. Convert to binary image (Process > Binary > Threshold)
16. Count cells (Analyze > Analyzed Particles… check Display results, Clear results table, Summarize).
17. Repeat counts with additional large squares to be sure that the cells are evenly distributed and to minimize stochastic error.  (The standard deviation is ~ sqrt(mean)).  

Note: An alternative to cropping the image is to instead photograph the fluorescent cells with a low level of brightfield illuminescense in order to visualize the lines of the hemocytometer together with the fluorescent cells.  Select the area of the large square for cell counts directly in ImageJ. 

Calculate the cell density
Cell density (cells/mL) = Average Cell Count (cells/large square)  *  10,000 (large squares/mL) * d.f. (dilution factor, if any)

Nucleus Extractiob From Single Mounted Tissue

Nucleus extractiob from single mounted tissue
 
 

1.  5   un   sect ion   of   paraffin-embedded   tissue   is mounted on a glass slide. Uncoated  or coated  slides (e.g. with aminotriethoxysilane or polylysine) can  be used .

 

2. The  mounted  section  is  dcparaffinized   in  I 00  ml xyle ne in a coplin  jar (twice. 5 min) and  reh ydrat ed in an  ethanol  series  ( 100. 90.  70 )for two min eachand 0.9'M,  NaCI solution ( twice. m i n). 

 

3. If necessa ry. undesira ble  parts  of  the  tissue can  be removed at  this  poi n t by scratchin g i t from the sl ide by  sca l pel.  This   ma y   be  hel pful w hen  anal yzing tumors  infiltrati ng normal tissue.

4.  After  that,  the  tissue  is  covered  wi th  proteinase  K solution (5 mg proteinase  K (Boeh ringer 745723), 50 M Tris-HCI  (pH 7.5), 20 0.5 M EDTA  (pH 7.0), 2 d 5 M NaCI,  ma k e up to  I   ml wi th fil tered double  distilled  water)  and  incubated   at  37°C  for I  h in a moist chamber.  Use of a coverslip shou ld be avoided.

5. Thereafter, t he protei nase  K  sol ut ion  t oget her wi th t he digested  tissue a nd released  nuclei  is collected  by pipette  a nd  passed  thro ugh a 55  pm  n yl on  mesh . Fl uid and  nuclei  will pass throu gh the mesh by force of gravity and  are collected  i n a 15 ml plastic t ube. Nuclei rema i ning in the mesh a re washed ou t by 4 ml I x PBS. passed  th rough  the mesh  a nd collected  in the 15 m l   plastic tu be.

 

6. Pellet the ext ract ed n uclei  by centrifuga tion (850 x g , 8 m i n) and remove the superna ta nt

 

7. Ta ke an  aliquot  of  the suspension  and  put  it on  a clean  and dry  test-sl ide by pi pett i n g the flu id  on t he sl ide surface.  A llow  t he d rop  to d ry ou t  on a 40°C wa rmin g plate a nd afterwards  at  room  tem perature overn i ght

 

8. St ore  the  rest  of  the suspension at  4°C  ove r nigh t and   pu t  i t  in  con ven ient   al iquots  on  sl ides  after evaluation  of the test-sl ide

 

9. Postfix t he test-sl i de in I 00 m l   forma l i n  buffer (i n I    x PBS)  in  a  cop!i n jar  for  l 0  min . Then   replace formali n-buffer i n  the copl in jar with I    x PBS. After 5 m i n o r incuba tion a t  room temperat ure. I     X   PBS is replaced  by d istilled  water.

10. Remove t he wa ter after  I   min.  per for m an et ha no l series (70. 90 a nd IOO'X,. 3 m i n each) to deh ydrate the test-sl id e  and  air  dry.  The success  of  the  nuclear ex traction  ca n   be a nal yzed  by ph ase contrast  l i g h t m icroscopy.  

 

11. Accord i ng  t o  the density  o f the  nuclei, on  the  test-sl ide, the  nucleus  suspension  stored  a t 4°C can  be d ist ribu ted o n  some  more slid es, which have to be treated  in the sa me way as t he  test-slid e before.  Thus,   from  one  section