The preparation of the samples for the observation in electron microscopy, requires the greatest precautions during the use of several solutions according to your protocol in various stages, the respect also of various determining and very important parameters like:
- the pH /Buffer,
- type of Buffer according to the sample,
- Osmolarity of the binding mixture
- temperature
- fixation duration.
The pH of the different cell compartments can vary, so you need to adjust the pH of your buffer solutions according to your studies. In general, in more than 50% of the cases the buffers remain close from 7 to 7.4 A buffer solution maintains approximately the same pH despite the addition of small amounts of an acid or base, or despite dilution.
phosphate or PB: A simple phosphate buffer is used ubiquitously in biological experiments, as it can be adapted to a variety of pH levels, including isotonic. This wide range is due to phosphoric acid having 3 dissociation constants- Phosphate Saline or PBS PBS has many uses because it is isotonic and non-toxic to most cells. These uses include diluting the substance and flushing the cells. PBS with EDTA is also used to release attached and clustered cells. this buffer is used in cell culture under controlled atmosphere in incubators with 5% CO2
- D-PBS: the The DPBS is Dulbecco’s PBS and also known as CMF-PBS (calcium-magnesium free, or Ca Mg Free). Ca Mg Free).
- cacodylates : Sodium cacodylate buffer [Na (CH3) 2 AsO2 – 3H2O] is an alternative to Sørensen phosphate buffer. It has good pH buffering capacity in the pH range 5.0-7.4. Cacodylate was introduced for electron microscopy applications by Sabatini et al. (1962) as a method to avoid adding additional phosphates to sample preparations. Mitochondria and other organelles can be damaged when exposed to the high concentrations of phosphates present in Sørensen’s buffers. In addition, cacodylate will not react with aldehyde fixatives as will amine-containing buffers (e.g. Tris). Its effectiveness in binding solutions may be the result of the inhibitory effect of arsenate metabolism rather than a special buffering capacity. Good’s buffers, or Good’s buffer-forming reagents, are a list of 20 buffer-forming chemicals selected for their suitability for use in biochemistry by Norman Good and his team from 1966 to 1980. These buffers are called “Good’s Buffers”. I will quote some of them used in Electron Microscopy.
- HEPES
- MOPS
- Tris , …
