LCM PROTOCOLS

Slide Sectioning

Paraffin blocks-

 For DNA analysis:
Special LCM processing schedule is followed.
The water bath is cleaned using RNAse Zap™, rinsed thoroughly with triple distilled water.
The water bath is filled with triple distilled water.
All instruments used are cleaned with 100% EtOH (brushes, forceps, pencil).
Sections not used are toweled off with kimwipes immediately.
A new blade is used for each block and 100% EtOH is used to wipe each blade while sectioning.
Gloves are worn at all times while sectioning and fresh gloves are put on every fifth block.
Talking is kept to a minimum while sectioning to avoid contamination of area.

 For RNA and Protein analysis:
Cryostat frozen sectioning used. Blade is wiped with 100% EtOH between blocks and dried.
Cryostat area and all instruments are cleaned with 100% EtOH.
All instruments used are cleaned with 100% EtOH (brushes, forceps, pencil).
A new blade is used for each block and 100% EtOH is used to wipe each blade while sectioning.
Gloves are worn at all times while sectioning and fresh gloves are put on every fifth block.
Talking is kept to a minimum while sectioning to avoid contamination of area.
 

 Staining of Paraffin Sections

Paraffin embedded tissue should be cut per guidelines and used as soon as possible for best results. This protocol has been used by several onsite investigators with good results for PCR.

1. Xylene (5 minutes)
2. Xylene (5 minutes)
3. 100% EtOH (30 seconds)
4. 100% EtOH (30 seconds)
5. 95% EtOH (30 seconds)
6. Distilled water (15 seconds) 
7. Mayer’s Hematoxylin ( 15 seconds) Filtered before every use!
8. .5% Lithium Carbonate in distilled water (30 seconds)
9. Distilled water (30 seconds)
10. 70% EtOH (30 seconds)
11. Eosin  (30 seconds) Filtered before every use! (alcoholic Eosin Y with Phloxine)
12. 95% EtOH (15 seconds)
13. 100% EtOH (30 seconds)
14. 100% EtOH (30 seconds)
15. Xylene (1 minute)
16. Xylene (1 minute)
 Air dry completely (2-4 minutes)

NIH protocol modified by NIEHS

  Staining of Frozen Sections

Frozen tissue should be cut per guidelines and stained as soon as possible for best results. This protocol has been used by several onsite investigators for RNA, DNA and Protein analysis.

6. Distilled water (15 seconds) 
7. Mayer’s Hematoxylin ( 15 seconds) Filtered before every use!
8. 1x Automation Buffer (30 seconds)
9. Distilled water (30 seconds)
10. 70% EtOH (30 seconds)
11. Eosin  (30 seconds) Filtered before every use! (alcoholic Eosin Y with Phloxine)
12. 95% EtOH (15 seconds)
13. 100% EtOH (30 seconds)
14. 100% EtOH (30 seconds)
15. Xylene (1 minute)
16. Xylene (1 minute)
 Air dry completely (2-4 minutes)

Mayer’s Hematoxylin  Cat#  MHS 16-500ml  Sigma
Eosin Y  Cat# 1B 425  CellPoint Scientific 25 gram
PhloxineB Cat# P-387  Daigger (Fisher)
1x Automation Buffer (Fisher)

NIH protocol modified by NIEHS

 Thumbnail Images

Procedure For Printing Thumbnails/Images From NIEHS LCM

1) Start ARC-100 software program and advance to LCM System User Selection
2) Highlight by clicking on the study name desired
3) Press Review Data
4) Choose appropriate folder(s)
5) Press Thumbnails
6) Scroll to put desired image(s) in view (if a single image is desired you can open it by double clicking on the image and proceding)
7) Double click the Shortcut to Snap32 icon on desktop.
8) Click OK
9) Press Ctrl+F11 keys. A blinking frame will appear around the thumbnails to be printed.
10) Click on blinking picture
11) Hypersnap finder with new thumbnail picture will open
12) Under File (menu) choose Print Setup to set parameters.
13) Control key +P to print images
14) Close and Exit all windows please.