Experimental study on concentration selection of immunohistochemical antibodies for retinal endoplasmic reticulum stress

Xiao-hong Chen; Wan-jiao Liang; Shi-shu Huang; Yan Sun; Xin Luo; Lu Lai; Zhao-sheng Chi; Mei-zhu Chen; Yun-peng Wang; Wei-ming Yan

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Experimental study on concentration selection of immunohistochemical antibodies for retinal endoplasmic reticulum stress

VernacularTitle:视网膜内质网应激免疫组织化学抗体浓度选择的实验研究
Author: Xiao-Hong CHEN ^{1
,

2
,

3} ; Wan-Jiao LIANG ^{1
,

2
,

3} ; Shi-Shu HUANG ^{1
,

2
,

3} ; Yan SUN ^{1
,

2
,

3} ; Xin LUO ^{1
,

2
,

3} ; Lu LAI ^{1
,

2
,

3} ; Zhao-Sheng CHI ^{1
,

2
,

3} ; Mei-Zhu CHEN ^{1
,

2
,

3} ; Yun-Peng WANG ^{1
,

2
,

3} ; Wei-Ming YAN ^{1
,

2
,

3}
Author Information

1. The 900th Hospital of Joint Logistic Support Force of Chinese PLA
2. Dongfang Hospital Affiliated to Xiamen University
3. Clinical Medical College of Fujian Medical University, Fuzhou 350025, Fujian Province, China
Publication Type:Journal Article
Keywords: retinitis pigmentosa; endoplasmic reticulum stress; retina; mice; immunohistochemical staining
From: International Eye Science 2023;23(1):32-38
CountryChina
Language:Chinese
Abstract: AIM: To explore the optimal concentration of endoplasmic reticulum stress immunohistochemical(IHC)staining antibody in mouse retinitis pigmentosa(RP)model, which provides the corresponding index detection method for studying the pathogenesis and intervention measures of RP.METHODS: Clean male C57BL/6J mice were intraperitoneally injected with N-methyl-N-nitrosourea(MNU, 60mg/kg)to prepare RP mouse model. Electroretinogram(ERG)and hematoxylin-eosin(HE)staining were performed on 7d after modeling to verify the successful modeling. The expression of endoplasmic reticulum stress-related proteins(IRE1, ATF6, PERK, GRP78, Caspase-12)was detected by IHC staining.RESULTS: The following proteins, including IRE1, ATF6, PERK, GRP78 and Caspase-12, were positively expressed in retina of RP mouse. The optimal concentrations of the above proteins were as follows: IRE1 antibody concentration was 1:1000, ATF6 antibody concentration was 1:500 and 1:1000(with no difference in positive expression, P>0.05), PERK antibody concentration was 1:1500, GRP78 antibody concentration was 1:200 and Caspase-12 antibody concentration was 1:100, the proteins were well expressed at the above concentrations, and the positive expressions of corresponding proteins were different from those of other antibody concentrations(P<0.05).CONCLUSION: The optimal concentrations for IHC staining in different proteins of mouse RP models were as follows: the concentrations of endoplasmic reticulum stress-related protein antibodies were 1:1000 in IRE1, 1:500 and 1:1000 in ATF6, 1:1500 in PERK, 1:200 in GRP78, and 1:100 in Caspase-12.