scholarly journals Protease Antigen Recovery Decreases the Specificity of Bromodeoxyuridine Detection In Formalin-fixed Tissue

1997 ◽  
Vol 45 (8) ◽  
pp. 1165-1170 ◽  
Author(s):  
Philip M. Bak ◽  
Ralph J. Panos
Keyword(s):  
Cellular Proliferation ◽  
Enzymatic Digestion ◽  
Antigen Retrieval ◽  
Alveolar Type ◽  
Positive Staining ◽  
Specific Staining ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Protease Digestion ◽  
Formalin Fixed

Incorporation of halogenated nucleotide analogues is often used to assess DNA synthesis and to quantitate cellular proliferation. Multiple antibodies have been developed to bromodeoxyuridine (BrdUrd) and it is the most frequently utilized substrate. Because the immunodetection of incorporated BrdUrd requires DNA denaturation or nuclease digestion, most of these antibodies are not reactive in tissues or cells fixed with crosslinking agents. Antigen retrieval techniques utilizing protease digestion restore BrdUrd antigenicity and permit the detection of BrdUrd in formalin-fixed tissue. However, during the development of a double label immunohistochemical protocol to quantitate proliferating alveolar Type II cells, we noted nucleus-specific staining in lung sections from animals that had not received BrdUrd. Therefore, we systematically analyzed the specificity of the immunohistochemical detection of incorporated BrdUrd in formalin-fixed tissue after protease digestion. Enzymatic antigen recovery diminished the specificity of the BrdUrd reaction product and caused false-positive staining with the BU-1, B44, and BR3 monoclonal antibodies. Staining was less prominent with Bu20a but was more specific. Protease antigen recovery may decrease the specificity of BrdUrd immunodetection. Appropriate controls are required when enzymatic digestion is used to detect incorporated BrdUrd in formalin-fixed tissue. The type and duration of fixation, antibody to BrdUrd, protease, and tissue may affect the specificity of the staining pattern. (J Histochem Cytochem 45:1165–1170, 1997)

Effects of antigen retrieval by microwave heating in formalin-fixed tissue sections on a broad panel of antibodies

1994 ◽  
Vol 102 (3) ◽  
pp. 165-172 ◽  
Author(s):  
R. von Wasielewski ◽  
M. Werner ◽  
M. Nolte ◽  
L. Wilkens ◽  
A. Georgii
Keyword(s):  
Microwave Heating ◽  
Antigen Retrieval ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Tissue Sections ◽  
Formalin Fixed

scholarly journals Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue.

1988 ◽  
Vol 36 (5) ◽  
pp. 511-514 ◽  
Author(s):  
Y Hayashi ◽  
M Koike ◽  
M Matsutani ◽  
T Hoshino
Keyword(s):  
Human Brain ◽  
Enzymatic Digestion ◽  
Fixation Time ◽  
Reaction Products ◽  
Human Brain Tumor ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.

Standardization of Immunohistochemistry Based on Antigen Retrieval Technique for Routine Formalin-fixed Tissue Sections

1998 ◽  
Vol 6 (2) ◽  
pp. 89-96 ◽  
Author(s):  
Shan-Rong Shi ◽  
Richard J. Cote ◽  
Benjaporn Chaiwun ◽  
Lillian L. Young ◽  
Yan Shi ◽  
...  
Keyword(s):  
Antigen Retrieval ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Tissue Sections ◽  
Retrieval Technique ◽  
Formalin Fixed

scholarly journals Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

1991 ◽  
Vol 39 (6) ◽  
pp. 741-748 ◽  
Author(s):  
S R Shi ◽  
M E Key ◽  
K L Kalra
Keyword(s):  
Polyclonal Antibodies ◽  
Antigen Retrieval ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Pre Treatment ◽  
Immunohistochemical Techniques ◽  
Formalin Fixed

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.

scholarly journals Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections.

1993 ◽  
Vol 41 (11) ◽  
pp. 1599-1604 ◽  
Author(s):  
S R Shi ◽  
B Chaiwun ◽  
L Young ◽  
R J Cote ◽  
C R Taylor
Keyword(s):  
Androgen Receptor ◽  
Enzymatic Digestion ◽  
Antigen Retrieval ◽  
Urea Solution ◽  
Paraffin Sections ◽  
Buffer Solution ◽  
Solution Ph ◽  
Citrate Buffer ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed

We developed a staining protocol for demonstration of androgen receptor (AR) in formalin-fixed, paraffin-embedded tissue sections. The method is based on the antigen retrieval microwave (MW) heating technique. Results are compared with different types of enzyme digestion pre-treatments. The strongest immunostaining signal and clearest background were obtained by MW heating of dewaxed paraffin sections in 5% urea or citrate buffer solution (pH 6); pure distilled water gave less consistent results. Enzymatic digestion with pepsin (0.05% in 2 N HCl) for 30 min at room temperature, or trypsin followed by pronase, or pronase digestion alone, also produced enhanced staining of AR in some cases, but there was more nonspecific background, and specific reactivity was less intense. The antigen retrieval MW method can be used to demonstrate AR epitope in prostate tissue after fixation in formalin for as long as 7 days. AR immunolocalization was also compared in frozen and paraffin sections processed from the same specimen of prostate carcinoma tissue and was found to be qualitatively and quantitatively similar. This study also provided new information concerning the basic principles of the antigen retrieval MW method that may be helpful in further development of this technique.

Sign in / Sign up

Export Citation Format

Share Document