【整理】如何降低石蜡冰冻切片，免疫荧光背景和自发荧光，国外专家的做法

做组织切片的免疫荧光染色，最头疼的问题之一就是背景荧光。自然情况下，很多有机物都有自发荧光的属性，更别提生物组织，所以不论是石蜡切片，还是冰冻切片一样存在背景荧光的问题。

解决的方法有很多：

1.使用共聚焦显微镜，成像会比一般荧光显微镜要好一些

2.使用纯光谱计算方法（compute pure spectrum，CPS）的CCD，如Nuance FX MSI ，可以通过复杂的计算扣掉背景质，当然这都是软件完成的。

3.以上方法各有优劣，1在于断层扫描，2在与成像质量更好，但是二者均很贵，对于普通实验室而言，最简单有效的方法就是通过化学的方法遮蔽自发荧光。这里举例，针对石蜡固定引起的自发荧光，可以考虑使用Sodium Borohydride ，具体的化学方法可以见附件，有更详细的说明，相信总有一个适合你的。

3.1 Protocols for reducing fixative-induced fluorescence

3.1.1 Sodium Borohydride

The use of these reagents is particularly suited to reduce the reversible Schiff's bases that areformed by the aldehyde-NH2 reaction and lead to autofluorescence, especially when using glutaraldehyde. If you can use paraformaldehyde for fixation, the reduction step is often unnecessary and autofluorescence is low. This material has a high potential for explosion and is very caustic.

The protocol was prepared by Jennifer Kramer and a similar procedure isdescribed by Beisker, et al. (Beisker et al. 1987).

1. Immediately before use, make up a 1 mg/ml solution of sodium borohydride in a physiological buffer such as PBS. The solution will be fizzy like carbonated water. Preparing this solution on ice and performing all subsequent incubations on ice has also been recommended.

2. Apply this solution immediately (while fizzing) to cells or tissue sections.For glutaraldehyde fixed cell monolayers incubate in the sodium borohydride solution for 4 minutes. Replace with fresh sodium borohydride solution for another 4 minutes.For paraformaldehyde fixed paraffin embedded 7 ?m sections incubate 3 times, 10 minutes each in sodium borohydride solution.