[Histonet] Ask for techenical help - about sections of tiny tissue.

[连 王]

Hello every Histonet - I could really need your help!
 
Our lab is currently obsevring pathlogical changes of aortic disease, especially 
elastic fiber and collagen in the background of abdominal aortic aneurysm (AAA). 
The aortic is from C57BL/6N mice.

First, I paid attention to the normal morphology of aortic artery.
But now the big problem is that the structure (mainly collagen and 
connective tissue) around the aortic artery couldn't be presevered. 
It seems to be destroyed, either in normal (with no stimulus) or AAA mice.
See my photo link:
  FIG 1 http://b212.photo.store.qq.com/psu?/6ebc69b4-17f2-43a6-8a04-6d96e913236e/ZMIqx1ix346XZIyxS1VvSWzGFiZVqs6Zq1x0C88Zzm4!/b/YQVfan6ScQAAYknHYn4wcQAA 
  FIG 2 http://b213.photo.store.qq.com/psu?/6ebc69b4-17f2-43a6-8a04-6d96e913236e/DAxmNEc.gpYU0RrrHx.IbhhjbH95LeEISm4c0BrlACM!/b/Ya5d.35gGwAAYriHBH.tGwAA 
  FIG 3 http://b212.photo.store.qq.com/psu?/6ebc69b4-17f2-43a6-8a04-6d96e913236e/4o43xMTRh6HzNzWacHUSmWN*4ZhH5.Y6RrqQl5SX7uI!/b/YcgpXn7ZcgAAYqHJYn5JcgAA 
  FIG 4 http://b212.photo.store.qq.com/psu?/6ebc69b4-17f2-43a6-8a04-6d96e913236e/sj62yPO5AmwjtA42o6asO11HxlKF4DQ8SyWMlTypyoc!/b/YYP0a36vcAAAYo1IYX4WcQAA 
 
I reviewed the orginal papers published in recent 10 years, and I am sure in 
our study conditions, these structures around the aortic ring should be intact.
 
See figure link(reprint from Ann Surg 2005;241: 92–101 and Atherosclerosis 2011;217: 350-357)
http://b211.photo.store.qq.com/psu?/6ebc69b4-17f2-43a6-8a04-6d96e913236e/X5QltJZABnkSKByT7E2K9SAcqt97uWhfgZBrmsyZ9w0!/b/YevLzn37cQAAYiAkx312cAAA 
and 
http://b210.photo.store.qq.com/psu?/6ebc69b4-17f2-43a6-8a04-6d96e913236e/mmpt1J8*7L*r0g6TBdCMZ3hk3n6KrOc9mKvXYmm4JdE!/b/YUXEOn0KnwAAYhUHLX0WnAAA 
 
 
In a word, this destruction seems to be due to technical procedure, but not the study-design itself.
 
The main steps are as follows:
1. Specimens were cut from sacrificed mice by minimally invasive scissors. It is impossible to destroy normal structure.
2. Tissue specimens were aortic artery between the renal arteries and the iliac bifurcation, which is about 8mm long and the diameter is 0.55mm.
   Given the tiny volumn, specimens were fixed by 10% neutral PBS-formalin for 2h. I have tried from 2h to 48h, the H&E stain result is similar.
3. Dehydration and clearing are automated by a mechanical processor. 
   Then the tissue is embedded with paraffin.
4. Embedded tissues were cut into 2-6 um sections with a microtome by a skilled technician. He has tried 2-6 um, and the H&E stain result is similar.
   Sections were floated on a room-temperature water bath, then transferred to a warm water bath (I have tried 40 or 50 degree centigrade bath, the H&E stain 
result is similar), then picked up on glass slides.

All procedures are performed in a Leica microsystem.

My questions are:
   1. Why the normal structure around aortic ring is destroyed?
   2. How to improve the procedure? 

Thank you in advance for all your suggestions!
 
Wang Lian, MD 
Medical School of Nanjing University
Hankou Road 22#
Nanjing, Jiangsu Province, China
e-mail: wanglianebox <@t> yahoo.com.cn