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WHAT IS DEEP MASKER?
WHAT IS DEEP MASKER?
Deep Masker is a validated, rapid, and objective deep learning-based algorithm to assess chord length from 2-D murine lung images. The algorithm was created by Jiantao Pu and Divay Chandra at the University of Pittsburgh.
Instructions (also available on the Deep Masker website): STRICT ADHERENCE TO THIS PROTOCOL IS ESSENTIAL
Instructions (also available on the Deep Masker website): STRICT ADHERENCE TO THIS PROTOCOL IS ESSENTIAL
Link to website:
Link to website:
New users will have to do a one-time registration when logging in for the first time.
New users will have to do a one-time registration when logging in for the first time.
Then scroll down and click the application mouse icon "A Deep Learning Based Fully Automated Tool to Assess Chord Length from Murine Lungs Images"
Then scroll down and click the application mouse icon "A Deep Learning Based Fully Automated Tool to Assess Chord Length from Murine Lungs Images"
Reference:
Reference:
Please cite the accompanying this paper when publishing the data generated by Deep Masker: https://www.atsjournals.org/doi/10.1165/rcmb.2023-0051MA
Introduction:
Introduction:
The following instructions describe how to harvest murine lung tissues, process and stain, and capture images in a manner compatible with Deep-Masker.
Inflating the lung:
Inflating the lung:
Once relevant exposures are complete, each mouse is sacrificed, tracheostomized, cannulated, and the lungs inflated with 10% buffered formalin at a constant pressure of 25 cm H2O for 5 minutes. The lungs are then fixed in formalin for 24 hours and subsequently embedded in paraffin. Midsagittal sections are obtained and stained with a modified Gill's stain as below. H and E staining is also compatible with the algorithm.
Gills staining protocol:
Gills staining protocol:
Obtain reagents:
- Xylene (EMD cat# UN1307)
- Ethanol (Pharmco cat# 111000200CSGL) 100%, 95%, 70%, 50%, 30%
- Gills stain (Sigma cat# GHS332)
- Harris Hematoxylin (Sigma cat# HHS32)
- Ammonium hydroxide solution (Fisher cat# A470-500)
- Permount mounting medium (Fisher cat# SP15-500)
Prepare reagents:
Ammonium hydroxide solution
- add 8 drops of 6N ammonium hydroxide to 250 mL diH2O
Staining solution
- mix 75 mL of Gills stain and 75 mL of Harris Hematoxylin
Procedure:
1. Turn on the oven to 60°C and place slides in a slide rack.
2. Place slide rack in oven for 30 min.
3. Wash with xylene 3 times; the first wash is for 2 min and the second and third washes are for 3 min each.
4. Rehydrate with ethanol as follows:
100% ethanol for 2 min
100% ethanol for 2 min
95% ethanol for 2 min
70% ethanol for 1 min
50% ethanol for 1 min
30% ethanol for 1 min
5. Wash with distilled water for 5 min.
6. Stain with staining solution for 16-24 hours.
7. Rinse with running distilled water until clear.
8. Wash with ammonium hydroxide solution for 5 min.
9. Wash with water for 30 seconds.
10. Dehydrate with ethanol as follows:
70% ethanol for 10 seconds
95% ethanol for 10 seconds
100% ethanol for 10 seconds
100% ethanol for 10 seconds
11. Wash with xylene 3 times for 1-2 min each.
12. Add a few drops of mounting medium to slide.
13. Place coverslip and blot excess mounting medium.
Capture images:
Capture greyscale images at 200x from 10 randomly selected fields per slide at 640*512 pixels.
Deep-masker will generate chord length in pixels. For pixel-to-micro conversion, check the resolution of your imaging system to see how many microns are there per pixel.
Using histowiz:
1. After gills staining send slides to Histowiz for whole slide scanning.
2. In the Histowiz viewer, set the magnification to 10x and save the image using the “screenshot” option. By default, the image will be PNG format and 1499 by 996 pixels in size with a 200-uM scale in the left bottom. To make this image analyzable in deep masker three things need to change: the size of the image in pixels, convert to greyscale, and change format to TIFF. Also, we need to know how many microns are there per pixel. We will use Photoshop to make these changes.
a. To convert pixels to microns, use the marquee tool to make a rectangle the same width as the 200-uM scale maker in the image and measure its width in pixels. I got 300 pixels. So, 300 pixels is 200 uM, or 1 pixel is 0.667 microns.
b. To change image size, choose the crop tool and select an area 640 by 512 pixels in size. Crop this area.
c. To remove color, go to the top menu: select image, then adjustments, then black and white (shortcut is alt+shift+control B). Set all the colors to -200%.
d. Now save this image in TIFF format using “save a copy”.
e. In the save as menu, uncheck the box for layers. Leave the TIFF options as they are.
f. Now upload into Deep-Masker and view results (leave image scale at the default value of 0.5).
g. Multiply pixels by 0.667 to convert the chord length measurement in deep masker from pixels to microns.
References:
References:
1. Hautamaki, R.D., Kobayashi, D.K., Senior, R.M. & Shapiro, S.D. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 277, 2002-2004 (1997).
2. Shapiro, S.D., et al. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice. Am J Pathol 163, 2329-2335 (2003).
3. Radder JE, Gregory AD, Leme AS, Cho MH, Chu Y, Kelly NJ, Bakke P, Gulsvik A, Litonjua AA, Sparrow D, Beaty TH, Crapo JD, Silverman EK, Zhang Y, Berndt A, Shapiro SD. Variable susceptibility to cigarette smoke-induced emphysema in 34 inbred strains of mice implicates Abi3bp in emphysema susceptibility. Am J Respir Cell Mol Biol. 57(3):367-375 (2017).
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