Antigen retrieval with protease digestion applied in immunohistochemical diagnosis of Alport syndrome.

BACKGROUND

Immunofluorescence (IF) staining of type IV collagen alpha chains on fresh frozen renal tissue is a convenient and accurate diagnosis technique for Alport syndrome (AS), which is restricted in the application with a non-frozen section. An alternative method for a paraffin-embedded section is needed in order to extend the application in various specimens. In this study, immunohistochemical staining of type IV collagen alpha chains on paraffin-embedded renal sections was investigated.

METHODS

Three antigen retrieval methods including autoclave heating, pepsin digestion and protease digestion were tried on paraffin-embedded renal sections from two X-linked male AS patients, two X-linked female AS patients and two autosomal recessive AS patients. Two patients with isolated haematuria were also involved. Normal portions of nephrectomized kidneys from two patients with renal tumours were used as controls. The immunohistochemical staining pattern of type IV collagen was compared with that of IF staining.

RESULTS

The results showed that both the autoclave heating method and protease digestion can be used for antigen retrieval for type IV collagen alpha chains. Compared to autoclave heating, protease digestion had the advantage of being less time consuming, with less renal sections being lost from the slides; it was the most optimal antigen retrieval method in this study. After protease digestion for 40 min, type IV collagen alpha3 and alpha5 chains showed a clear and proper distribution pattern in AS patients, haematuria patients and controls, which is the same as that of IF staining.

CONCLUSIONS

Compared to the autoclave heating method, protease antigen retrieval is more convenient and effective and can be used to restore type IV collagen alpha chains on paraffin-embedded renal sections for the diagnosis of AS.