Fixatives paraformaldehyde

Preparing and using fixatives containing formaldehyde must be done in a fume hood. 1. Paraformaldehyde Stock Solution (15%) makes 200 ml 

In a 500 ml Erlenmeyer beaker with 100 ml of distilled water, add 30 g of paraformaldehyde powder. 

In a fume hood, place beaker on hot plate, add stir bar, and warm to 70°C. Add drop-wise 1 M NaOH slowly and the solution will begin to clear. Add up to 1 ml or until the solution does not clear further. This solution will not be totally clear. 

Let it cool to room temperature in the hood. Add 99 ml of distilled water. Filter through the Whatman No. 1 filter paper in the hood. The solution should be clear. 

Phosphate buffered saline (PBS) add the following in order to a volumetric flask beginning with 800 ml of ddH20 

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Gill et al.21 Archival FFPE spinal cord tissue both paraformaldehyde-fixed frozen rat spinal cord tissue and paraffin-embedded same tissue To establish an optimal protocol for detection of low-abundance protein (NeuN) in human spinal cord FFPE tissue sections, testing three AR solutions of pH 6, alkaline, and acidic buffer, with three heating conditions 95,100, and 105°C Heating FFPE tissue sections in an alkaline buffer yields most effective AR-IHC staining results. 

AR by Heating En Bloc for Paraformaldehyde -Fixed Frozen Tissue... 

Figure 6. Cytoskeletal proteins in nucleus. HeLa cells were immunostained with anti-keratin 8 or anti-keratin 18 antibodies, (a) Paraformaldehyde-fixed cells, (b) Cells treated with detergent before the fixation. (Scale bars 10pm)...

Paraformaldehyde fixes by crosslinking surface proteins. Ethanol and methanol work through precipitation of membrane components. Triton X-100 and Tween-20 permeabilize membranes. Other permeabilizers are saponin (0.1-10% [v/v] in PBSG), L-lysophosphatidylcholine, and n-octyl-P-D-glucopyranoside (1-10 pg/mL in water). 

Cut formalin- or paraformaldehyde-fixed paraffin-embedded tissne into 4-5-pm-thick sections on a microtome. 

Free-floating sections (40 xm) of paraformaldehyde-fixed tissues are rinsed three times for 5 min each in 0.1 M sodium phosphate buffer (pH 7.4) (Jiao et al., 1999). They are transferred to 10-15 mM sodium citrate buffer (pH 8.5-9.0) preheated in a water bath kept in a conventional oven at 80°C for 30 min. The sections are allowed to remain in this buffer for 30 min to cool to room temperature. Following rinsing three times for 5 min each in the same buffer, the sections are treated by immersion in 0.3-3% nonfat dry milk in 0.1 % sodium azide for 30-60 min. The sections are then incubated in the primary antibody, diluted with a mixture of 0.3% Triton X-100, 0.01% sodium azide, 0.1 M sodium phosphate buffer (pH 7.4) (PBX), and 5% normal horse serum for 72 hr at 4°C under constant agitation. 

Hannah, M. J., Weiss, U., and Huttner, W. B. 1998. Differential extraction of proteins from paraformaldehyde-fixed cells Lessons from synaptophysin and other membrane proteins. Methods Enzymol. 26 170-181. 

Peranen, J., Rikkonen, M., and Kaariainen, L. 1993. A method for exposing hidden antigenic sites in paraformaldehyde-fixed cultured cells, applied to initially unreactive antibodies. J. Histochem. Cytochem. 41 441-454. 

McLean IW, Nakane PK (1974) Periodate-lysine-paraformaldehyde fixative A new fixative for immunoelectron microscopy. J Histochem Cytochem 22 1077-1083... 

To allow efficient extraction of intact mRNA, it is recommended that alcohol fixatives be used in lieu of cross-linking agents such as paraformaldehyde. In our experience protocols that have been developed for studying paraformaldehyde-fixed paraffin-embedded archival specimens in tandem with RNA amplifications produce low yields of RNA from microdissected brain regions and picked cells. Fixation of tissue with methanol or ethanol preserves immu-noreactivity and mRNA in many cases. Accordingly, we immerse... 

Place the tissue in 4% paraformaldehyde fixative cut the tissue into small pieces. 

Incubate the pieces in the 4% paraformaldehyde fixative for 2-3 h with agitation. 

Performing immunohistochemistry on these rehydrated paraffin sections frequently leads to poor results. In contrast, the same antibodies on paraformaldehyde-fixed and cryostat sections will give good results. The issue with formalin-fixed and paraffin-embedded tissue is that the exposure to formalin and dehydration alters the epitopes in the tissue. As a result, formalin-fixed and paraffin-embedded tissues need additional processing methods, known as epitope retrieval or antigen retrieval. Done before immunohistochemistry, epitope retrieval involves heating the sections in buffer with either an acid or base to allow the antibody to recognize the epitope. Also, the exact process of epitope retrieval can be different for individual antibodies. There are numerous papers and books on epitope retrieval and how to apply the method. 

Archival FFPE spinal cord tissue both paraformaldehyde-fixed frozen rat spinal cord tissue and paraffin-embedded same tissue... 

Paraformaldehyde fixative 4% (w/v) paraformaldehyde in phosphate buffer (see item 1). Dissolve 80 g of paraformaldehyde in 1000 mL of distilled water while heating on a stir plate to about 60 C under a well-ventilated fume hood. Add IM NaOH dropwise until the solution is clear (to completely dissolve the aldehyde) Cool on ice, then mix this 8% (w/v) paraformaldehyde solution with an equal volume of 0,2M sodium phosphate buffer (see item 1) to obtain the final fixative. Adjust the pH to 7.2. This fixative can be aliquoted and stored frozen in vials After thawing, mix the solution well and check the pH. 

Paraformaldehyde fixative Dilute paraformaldehyde fixative (Section 2.1., item 5) with an equal volume of phosphate buffer (Section 2.1., item 1). 

Perfuse with paraformaldehyde fixative As formaldehyde is a toxic and hazardous chemical, perform perfusion in a well-ventilated area or preferably, under a fume hood (see Note 18). 

Immerse the tissue overnight (16 h) at 4- °C in freshly prepared paraformaldehyde fixative (see Notes 19 and 20). 

Immunocytochemical Detection of Amino Acid Neurotransmitters in Paraformaldehyde-Fixed Tissues... 

Paraformaldehyde fixative (see Note 1) 4% (w/v) paraformaldehyde (extra pure, Merck, Darmstadt, Germany) in 0.1 Af phosphate buffer, pH 7.2-7.3.. Add 16 g of paraformaldehyde to 100 mL of distilled water and warm to OO C, with stirring. Slowly add drops of sodium hydroxide solution until the paraformaldehyde dissolves (depolymerizes) completely. Add distilled water, bringing the volume to 200 mL. To this add 200 mL of phosphate buffer B. The fixative must be prepared in and only used in a fiime hood because it is toxic by inhalation and skin contact. 

Fig. 4. A summary of stq)s involved in the immunocytochemical detection of ammo acids in paraformaldehyde-fixed tissues. Details are given in die protocols and Notes. SABHRP = str tavidm-biotm-horseradish peroxidase conqilex DAB = 3,3 diaminobenzidine...

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