Effect of formalin pigment removal on peroxidase-antiperoxidase immunoperoxidase technique.

In immunoperoxidase preparations formalin pigment (acid formaldehyde haematein) may be difficult to distinguish from the brown staining of the 3,3'-diaminobenzidine (DAB) reaction product. The pigment, produced when formalin is in contact with blood rich tissues, takes the form of dark brown microcrystals that have the ability to rotate the plane of polarised light.' Even though formalin pigment can be differentiated by this means, the technique may be cumbersome and tedious. Several methods for the removal of formalin pigment have been described. These have been performed in the current study, both before and after treatment in immunoperoxidase sequences. Specimens of normal splenic tissue were sampled from necropsies. The tissue was fixed in 10% formol saline and then routinely processed and embedded in paraffin wax. Sections were then cut at 2-3 pm. The following techniques for the removal of formalin pigment were carried out, both before and after the immunoperoxidase staining sequence: I Saturated alcoholic picric acid followed by 30 minutes running tap water (15 minutes). 2 One per cent sodium hydroxide in 70% ethanol followed by five minutes running tap water (10 minutes). 3 Ten volumes hydrogen peroxidase followed by five minutes running tap water (five hours). 4 Concentrated nitric acid followed by 15 minutes running tap water (one hour). 5 Five per cent chromium trioxide followed by 15 minutes running tap water (30 minutes). 6 Five per cent potassium permanganate (two minutes), followed by 5% oxalic acid, (two minutes), followed by five minutes running tap water. These methods are standard techniques.' IMMUNOPEROXIDASE METHOD After treatment with 0-1% trypsin in calcium chloride solution for 20 minutes the endogenous peroxidase activity was blocked using a solution of 0 3% hydrogen peroxidase in methanol. Non-specific background staining was minimised by the application of normal swine serum at a titre of 1/5. After 10 minutes the excess normal swine serum was discarded and optimally diluted antisera directed against K and A' immunoglobulin light chains were overlaid for 20 minutes. (The antisera used were obtained from Dako, United Kingdom). The sections were washed for five minutes, after which swine-antirabbit serum was applied for 20 minutes at a titre of 1/50. After a further wash rabbit peroxidase antiperoxidase (PAP) complex (1:50) was overlaid. The peroxidase was demonstrated by a 3,3'-diaminobenzidine reaction. To diminish the possibility that our results were true only for sequences for light chains sections were also stained for the following molecules: cathepsin G, …