Paraformaldehyde protein-antibodies

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)...

Figure 7. A series of monoclonal antibodies raised against nuclear proteins. HeLa cells fixed with 4% paraformaldehyde were immunostained with monoclonal antibodies raised against nuclear proteins. The intracellular localization of these antigens are (a) dot inside the nucleolus, (b) whole nucleolus, (c) nuclear foci, (d) nucleoplasm, (e) the edge of the nucleus, (f) cytoplasm, (g) cytoskeleton, (h) plasma membrane, (i) mitochondria, (j) nucleus and cytoplasm, (k) nucleus and the paranuclear structure, and (1) paranuclear structure and nucleoplasm...

Embedding the tissue with standard hydrophobic epoxy resins will not allow antibody penetration, even to the edge of the tissue section. Two approaches are used to circumvent this problem. First, tissue sections with a standard resin are treated with 10% H2O2 to etch the epoxy section on the sections cut surface allowing antibodies to contact proteins in the section. Fixation can be with paraformaldehyde or... 

Figure 2 Neonatal rabbit lung slice preparation (200 pm) fixed in 2% paraformaldehyde, immunostained with antibody against synaptic vesicle protein 2 (SV2), and examined by confocal microscopy. Typical NEBs witbin the airway mucosa with adjacent submucosal nerves (arrows) some reaching the NEB cell clusters (x250). Insert Higher magnification of NEB with both NEB cell cytoplasm and intracorpuscular innervation (arrow) labeled by SV2 antibody (x650).

One of the two protocols presented here has been adjusted to process paraformaldehyde-fixed tissues while the second for tissues that require glutaraldehyde fixation since the primary antibodies utilized are developed with immunogens obtained by coupling small molecules (e.g., neurotransmitters) with carrier proteins (for details see AO). The techniques described herein are based on those... 

Using the first protocol (frozen sections), samples have been fixed in paraformaldehyde since haptens used for production of the antibodies (i.e., anti-GABA and anti-5-HT) have been conjugated to BSA (carrier protein) via paraformaldehyde. Figure 1 Top shows a detail of the brachial lobe (part of the subesophageal mass) with cell bodies and fibers positive to 5-HT immunoreactivity. 

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