Immunolabeling recovery in archival, post-mortem, human brain tissue using modified antigen retrieval and the catalyzed signal amplification system.
Human brain tissue is a valuable source of material for research. It is often stored indefinitely in formalin at room temperature which may weaken the immunolabeling with formalin-sensitive antibodies. The present study found that a novel protocol that combined citrate and formic acid pre-treatments with the catalyzed signal amplification (CSA) system was able to recover the lost or weakened immunolabeling with the formalin-sensitive antibodies, anti-CD34, anti-caveolin, anti-P-glycoprotein, anti-neuronal nuclei, anti-parvalbumin, anti-human leukocyte antigen, anti-CD45, anti-CD68 and anti-connexin 43, in post-mortem, human brain tissue that was stored in formalin for up to 10 years at room temperature. Recovered immunolabeling in long-fixed tissue resembled immunolabeling observed in tissue that was fixed for a shorter duration between 6 and 49 days. The findings from this study highlight the importance of testing antibodies for formalin fixation effect prior to studies, especially if long-fixed tissue is used, to enable immunolabeling to be more accurately interpreted. Importantly, this study provides a method of overcoming formalin-masking of antigens in long-fixed human tissue, thus allowing essential immunohistochemical studies to be undertaken using precious human tissue.
Liu
Joan Y W
JY
Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology and National Hospital for Neurology and Neurosurgery, 33 Queen Square, London WC1N 3BG, UK. j.liu@ion.ucl.ac.uk
Martinian
Lillian
L
Thom
Maria
M
Sisodiya
Sanjay M
SM
D000328:Adult; D000368:Aged; D000941:Antigens / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D019343:Citric Acid; D004827:Epilepsy / Q000378:metabolism; D005260:Female; D005557:Formaldehyde; D005561:Formates; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D008875:Middle Aged; D013696:Temperature; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*
D000941:Antigens; D005561:Formates; C030544:formic acid; D005557:Formaldehyde; D019343:Citric Acid