SiriusRedstaining天狼星红染色方法
1、来自丁香园
Sirius red苦陈酸染色法
[试剂配制]
(1)天狼星红饱和苦昧酸液 O(5,天狼星红10ml，苦味酸饱和液90ml。
(2)天青石蓝液 天青石蓝B1(25g(铁明矾1(25g，蒸馏水250ml。溶解煮沸、待冷却过
滤
加入甘油30ml，然后再加入浓盐酸0(5ml。
[染色步骤]
(1)中性甲醛液固定组织，石蜡切片，常规脱蜡至水。
(2)人大青石蓝液染5一lOmin。
(3)蒸馏水洗3次。
(4)天狼星红饱和苦昧酸浓染15-30min
(5)无水乙醇直接分化与脱水。
二甲苯透明，中性树胶封固。
[注意事项]
(1)细胞核里染色可以用Harris苏木素染色液谈染。
(2)染色封固后的切片，须及时用偏光显微镜进行观察和照相已保持鲜艳的色彩
注:在偏光显微镜下可以观察到四种类型的胶原纤维。
l型胶原纤维:紧密排列，显示很强的双折光性，呈黄色或红色的纤维o
ll型胶原纤维:显示弱的双折光，呈多种色彩的疏松网状分布。
皿型胶原纤维:显示弱的双折光，呈绿色的细纤维。
lv型胶原纤维:显示弱的双折光的基膜，呈淡黄色。
2、来自网页
Sirius Red Staining Protocol for Collagen
John A. Kiernan
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
NovaUltra Special Stain Kits
Description: It's one of the best understood techniques of collagen
histochemistry. Technical details follow, and are followed by some
comments and a few references. You should come to grips with the
theory, advantages and limitations of this method before using it on a
large scale. Picro-sirius red method (after Puchtler et al., 1973; Junqueira et al., 1979). Step 4 is an addition that prevents the loss of dye that happens if the stained ctions are washed in water.
Fixation: Fixation is not critical, The method is most frequently ud on paraffin ctions of objects fixed adequately (at least 24 hours but ideally 1 or 2 weeks) in a neutral buffered formaldehyde solution. This protocol has not been tested on frozen ctions.
Solutions and Reagents:
Picro-sirius Red Solution
Sirius red F3B (C.I. 35782) ------------------------- 0.5 g
Saturated aqueous solution of picric acid ---------500 ml
(Keeps for at least 3 years and can be ud many times)
Sirius Red is available from Sigma-Aldrich under the name of "Direct Red 80" Cat#365548 or Cat#43665. Saturated aqueous solution of picric acid (1.3% in water) is also available from Sigma, Cat# P6744-1GA.
Acidified Water
Add 5 ml acetic acid (glacial) to 1 liter of water (tap or distilled).
Weigert's haematoxylin
Procedure:
1. De-wax and hydrate paraffin ctions.
2. Stain nuclei with Weigert's haematoxylin for 8 minutes, and then wash the slides for 10 minutes in running tap water).
3. Stain in picro-sirius red for one hour (This gives near-equilibrium staining, which does not increa with longer times. Shorter times should not be ud, even if the colors look OK.)
4. Wash in two changes of acidified water.
5. Physically remove most of the water from the slides by vigorous shaking.
5. Dehydrate in three changes of 100% ethanol.
6. Clear in xylene and mount in a resinous medium.
Results:
In bright-field microscopy collagen is red on a pale yellow background. (Nuclei, if stained, are ideally black but may often be grey or brown. The long time in picro-sirius red caus appreciable de-staining of the nuclei. This is not a problem with traditional van Gieson or with picro-aniline blue, with their 1-minute staining times.)
When examined through crosd polars the larger collagen fibers are bright yellow or orange, and the thinner ones, including reticular fibers, are green. According to Junqueira et al. (1979) the birefringence is highly specific for collagen. A few materials, including Type 4 collagen in bament membranes, keratohyaline granules and some types of mucus, are stained red but are not birefringent. It is necessary to rotate the slide in order to e all the fibres, becau in any single orientation the birefringence of some fibres will be extinguished. This minor inconvenience can be circumvented by equipping the microscope for u with circularly rather than plane polarized light (Whittaker et al., 1994; Whittaker, 1995), but then you don't get a completely black background. Comments and References:
Although this method is technically very easy, it is important for the person doing it and (if it's someone el) the person using the stained slides, to know what it does and how it works. Even without a polarizing
microscope, picro-sirius red shows things like reticular fibres and the basal laminae of cer
ebral capillaries, which are misd by van Gieson and may be obscured by mass of other stained details in trichrome methods (Mallory, Masson, Heidenhain etc).
To the best of my knowledge, most urs of picro-sirius red are doing rearch that exploits the enhancement by sirius red of the birefringence of collagen fibres, which is largely due to co-aligned molecules of Type I collagen. It is also ud to stain amyloid.
If you are using only polarized light it does not matter if you lo the "yellow background" of picric acid staining. If you u picro-sirius red as a "better" van Gieson and want to keep the yellow cytoplasm, be hasty with the dehydrating - even more so than with the original van Gieson method.
