Golgi–Cox method requires impregnation of fresh unfixed brain blocks. However, if PFA-fixed or formaldehyde-fixed tissues have to be used, some special treatments for fixed tissues should be performed to get good results.

1. The animal must be completely perfused with buffer and fixative. Perfusion should be performed within five minutes after death of the animal. Once tissues are removed, transfer them into the fixative as soon as possible. Fixed and sucrose solution treated tissues can be stored at -70°C for a long time.

2. Before Golgi-Cox staining, the fixed tissue needs to be placed under running tap water for at least 24 hours.

3. Transfer tissue into distilled water and store at room temperature in the dark for another 24 hours.

4. Transfer tissue into the impregnation solution and follow Tissue Preparation protocol from step 4 like unfixed Golgi-Cox brain tissues (Hito Golgi-Cox OptimStain™ Kit User Manual Page 6 – Standard Protocol or Page 8 – Vibratome Protocol) After the treatment, staining quality with fixed tissue is satisfactory for demonstrating dendritic branching pattern and dendritic spines. Glia cells can also be stained. Compared to the fresh tissues, PFA-fixed tissues or formaldehyde-fixed tissues have stained glia cells which contribute to higher background. Though we do not recommend using fixed tissues, our HITO GOLGI-COX OPTIMSTAIN™ KIT provides an alternative approach to do research with fixed pathological samples.

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