There are many buffers used for Heat-Induced Epitope Retrieval (HIER) process to unmask antigen determinant foldings caused by “cross-linking” of formalin fixation in the tissues. Some buffers may be good for retrieval of certain antigens, while others may not be suitable for the same antigens. By optimizing the chemical contents and pH readings applied in available antigen retrieval buffers, we have formulated a universal buffer for effective unmasking of various antigen determinants. This universal antigen retrieval buffer also provides very gentle pH and buffering conditions to maintain tissue section integrity at high temperatures.