wsqin326

福尔马林固定时间较久，请问铬化处理如何进行？多谢！

ylzhao

[这个贴子最后由ylzhao在 2004/10/09 10:28pm 编辑]

根据研究的需要和组织大小确定固定时间，常规H.E染色固定梢长一点没有什么关系。如果做免疫组化，固定时间不宜太长，不然会影响结果。10X10X3mm大小的组织10%缓冲福尔马林中固定6-24小时对免疫组化不会有影响。缓冲福尔马林固定组织的速度大约1～4小时/mm，大标本应延长固定时间。一般固定后及时进行组织脱水处理，如果不能及进行组织脱水处理。固定时间完成后可以将组织转移到70%乙醇中适当延缓处理。组织转移到70%乙醇中的目的为了解除甲醛继续对组织固定。
三色也用到铬化处理，具体染色方法上都有。另外所指的铬化处理，是不是用于载玻片和玻璃器皿清洁的铬硫酸液。如果是我说的“铬化处理”。很多书里都可以查到它的配方和用途。我们通常把它称为清洁液或铬硫酸清洁液。成分是重铬酸钾、硫酸、蒸馏水或自来水。详细参考下面网页。
http://www.childrenshospital.org/silverman/protocols/mrc/chromic.pdf

ylzhao

对不起 我没有看清楚主题，回答的不准确，应该是脱色素吧，有时间我再回复。

ylzhao

[这个贴子最后由ylzhao在 2004/10/10 01:12pm 编辑]

三色染色有时候用ZENKER固定剂或含有重铬酸钾液体做媒染剂。还有一种六氨银染色用铬酸处理。你是做三色染色还是做别的什么染色。
下面是重铬酸钾在染色中的应用参考：
http://members.pgonline.com/~bryand/StainsFile/stain/conektv/tri_milligan.htm
有关铬酸在染色中的应用
Grocott's Silver-Methenamine Method for Fungus
Fixation: 10% Neutral buffered formalin
Technique: Paraffin sections cut at 4 microns
Control: Tissue containing fungus
Solutions: 5% aqueous silver nitrate, with 3% methenamine
10% Chromic Acid Solution
Chromium trioxide 5.0 g
Distilled water 50.0 ml
1% Sodium Bisulfite Solution
Sodium bisulfite 0.5 g
Distilled water 50.0 ml
2% Sodium Thiosulfate
Sodium thiosulfate 2.5 g
Distilled water 50.0 ml
Methenamine-Silver Nitrate Solution (Stock)
5% Silver nitrate 5.0 ml
3% Methenamine 100.0 ml
A white precipitate forms but immediately dissolves on shaking. Clear solutions remain usable for months. Store in refrigerator.
Methenamine-Silver Nitrate Solution (working)
Methenamine-silver nitrate solution (stock) 25.0 ml
Distilled water 25.0 ml
5% Borax 2.0 ml
Make this solution fresh each time.
0.1% Gold Chloride Solution
1% Gold Chloride solution 5.0 ml
Distilled water 45.0 ml
This solution may be used until brown precipitate appears and the solution is cloudy.
1% Light Green Solution (stock)
Light green SF yellowish 1.0 g
Distilled water 100.0 ml
Glacial acetic acid 0.2 ml
Thymol 2.0 grains
1% Light Green Solution (working)
Light green, stock 10.0 ml
Distilled water 40.0 ml
Staining Procedure:
Attach to automatic stainer
Deparaffinize sections and hydrate to distilled water. Slides previously stained with most other stains may be used by removing coverslips and running through xylene and alcohols to water. Subsequent chromic acid treatment will remove any remaining stain.
Oxidize in fresh 10% chromic acid solution for 10 minutes at room temperature. (Note: this step may also be performed in a vented laboratory microwave set at 60 C for 90 seconds).
Rinse in distilled water for a few seconds.
Rinse until clear in 1% sodium bisulfite to remove any residual chromic acid.
Rinse in distilled water 5 times.
Place slides in Silver Methenamine Solution and microwave for 45 seconds. Let slides set for 2 minutes. Examine microscopically. Fungi should be dark brown. If staining is not complete microwave again in the hot working solution for 10 seconds. Re-examine slides. Repeat this step until the desired staining intensity is reached.
Rinse in 5 changes of distilled water.
Tone in 0.1% gold chloride solution until sections are gray-lavender, 1 minute.
Rinse in distilled water
Remove unreduced silver with 2% thiosulfate (hypo) for 1 minute.
Wash thoroughly in tap water.
Counterstain with working light green solution for 15 seconds.
Dehydrate, clear and mount with xylene soluble media.
Results:
Fungi - sharply delineated black
Mucin - taupe to dark gray
Inner parts of mycelia & hyphae - old rose
Background - pale green
Adapted for the Jefferson Regional Medical Center Histo-Path Laboratory
References
Grocott, R.G.: American Journal of Clinical Pathology, 25: pp 975-979, 1955.