Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

摘要

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

摘要

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A "neurochemical signature" to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.

Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.

Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

引言

Proteins and mRNAs that neurochemically define subpopulations of neurons are commonly identified with a combination of immunohistochemistry (IHC) and/or in situ hybridization (ISH), respectively. Combining ISH with IHC techniques facilitates the characterization of colocalization patterns unique to functional neurons (neurochemical coding) by maximizing multiplex labelling capacity.

Fluorescent ISH (FISH) methods, including RNAscope, have higher sensitivity and specificity compared to earlier RNA detection methods such as radioactive ISH and non-radioactive chromogenic ISH. FISH enables visualization of single mRNA transcripts as p....

方案

A summary of tissue pre-processing steps may be found in Figure 1. All procedures were carried out in compliance with the Animal Care and Ethics Committee of the University of New South Wales in accordance with the guidelines for the use and care of animals for scientific purposes (Australian National Health and Medical Research Council).

1. Sample preparation of fresh frozen brain tissue

1. Transcardial Perfusion
   1. Prepare heparinized (2500 U/L) 0.1 M phosphate buffer (PB), pH 7.5. Make dry ice ethanol slurry by mixing dry ice with ethanol. This will have a temperature of approximately −72 °....

结果

Here, we outline a method for combining multiplex FISH with fluorescent IHC to localize mRNA expression for GalR1 and GlyT2 using fresh-frozen and paraformaldehyde fixed tissues respectively in the mouse NTS. A pipeline of the tissue processing, FISH and IHC procedures described in the methods is displayed in Figure 1 and Figure 2. Table 1 provides a summary of the FISH probe and antibody combinations used in each figure.

讨论

In the neurosciences, FISH and IHC are routinely used to investigate the spatial organization and functional significance of mRNA or proteins within neuronal subpopulations. The protocol described in this study enhances the capacity for simultaneous detection of mRNAs and proteins in brain sections. Our combined multiplex FISH-IHC assay enabled phenotypic identification of distinct neuronal subpopulations in the NTS in both fresh frozen and fixed brain preparations. FISH-IHC in fixed frozen tissue preparations produced r.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
ANIMALS
C57BL/6 小鼠	澳大利亚生物资源，Moss Vale	MGI：2159769
Phox2b-eGFP 小鼠	澳大利亚生物资源，Moss Vale	MGI：5776545
试剂
氰基丙烯酸酯	乐铁矿
乙二醇	Sigma-Aldrich	324558
肝素-钠	Clifford Hallam Healthcare	1070760	咨询当地兽医供应商或药房。
Lethabarb（五巴比妥钠）安乐死注射液	Virbac （Australia） Pty Ltd	N/A	咨询兽医，了解当地有关五巴比妥钠的制药法规
级琼脂糖粉末	Sigma Aldrich	5077
OCT 化合物，118mL	Scigen Ltd	4586
多聚甲醛，颗粒，95%	Sigma-Aldrich	441244-1KG
聚乙烯吡咯烷酮，平均分子量 40,000 （PVP-40）	Sigma-Aldrich	PVP40
ProLong Gold 抗淬灭封片剂	Invitrogen	P36930	含或不含 DAPI
RNAscope 多重荧光试剂盒（高达 3 重能力）	Advanced Cell Diagnostics， Inc. （ACD Bio	ADV320850	包括 50x 洗涤缓冲液和蛋白酶 III
RNase Away	Thermo-Fisher Scientific	7003
Tris（羟甲基）氨甲烷	Sigma-Aldrich	252859
Tween-20，用于分子生物学	Sigma-Aldrich	P9416
EQUIPMENT
台式培养箱	Thermoline 科学微型培养箱	型号：TEI-13G
脑基质，小鼠，30g 成年，冠状，1 毫米	Ted Pella	15050
低温恒温器	Leica	CM1950
拉针（23 英寸规格）	BD	0288U07
疏水屏障笔	Vector labs	H-4000
Kimtech Science Kimwipes 精致任务湿巾	Kimberley Clark Professional	34120
奥林巴斯 BX51	奥林巴斯	BX-51
蠕动泵	Coleparmer Masterflex	L/S 系列
Retiga 2000R 数码相机	QImaging	RET-2000R-F-CLR	彩色相机
SuperFrost Plus 载玻片（白色）	Thermo-Fisher Scientific	4951PLUS4
振动切片机（振动切片机）	Leica	VT1200S
Whatman 定性滤纸，1 级，直径 110 毫米	Merck	WHA1001110
软件
CorelDRAW	Corel Corporation	版本 7
斐济（ImageJ Distribution）	开源/GNU 通用公共许可证 （GPL）	N/A	ImageJ 2.x：Rueden， C. T.;辛德林 J. &Hiner， M. C. et al. （2017），"ImageJ2：用于下一代科学图像数据的 ImageJ"，BMC 生物信息学 18：529，PMID 29187165，doi：10.1186/s12859-017-1934-z  斐济：Schindelin， J.;Arganda-Carreras， I. &Frise， E. et al. （2012），"斐济：生物图像分析的开源平台"，Nature methods 9（7）：676-682，PMID 22743772，doi：10.1038/nmeth.2019
<强>一抗
抗酪氨酸羟化酶抗体	Millipore Sigma	AB1542	绵羊多克隆抗体（1：1000 稀释），RRID：AB_90755
抗酪氨酸羟化酶抗体，克隆 LNC1	Millipore Sigma	MAB318	小鼠单克隆抗体（1：1000 稀释），RRID：AB_2201528
抗泡状乙酰胆碱转运蛋白 （VAchT） 抗体	Sigma-Aldrich	ABN100	山羊多克隆抗体（1：1000 稀释），RRID：AB_2630394
GFP 抗体	Novus Biologicals	NB600-308	兔多克隆抗体（1：1000 稀释），RRID：AB_10003058
Phox2b 抗体 （B-11）	Santa Cruz Biotechnology	sc-376997	小鼠单克隆抗体（1：1000 稀释），RRID：AB_2813765
<强>二抗
Alexa Fluor 488 AffiniPure 驴抗兔 IgG （H+L） （min x Bov， ck， Gt， GP， sy hms， hrs， 胡， ms， rat， shp sr prot）	Jackson ImmunoResearch	711-545-152	驴抗兔（1：400 稀释），RRID：AB_2313584
AMCA AffiniPure 驴抗绵羊 IgG （H+L）（min X Ck、GP、Sy Hms、Hrs、胡、Ms、Rb、Rat Sr Prot）	Jackson ImmunoResearch	713-155-147	驴抗绵羊（1：400 稀释），RRID：AB_AB_2340725
Cy5 AffiniPure 驴抗羊 IgG （H+L）（min X Ck、GP、Sy Hms、Hrs、胡、 Ms， Rb， Rat Sr Prot）	Jackson ImmunoResearch	705-175-147	驴抗山羊（1：400 稀释），RRID：AB_2340415
Cy5 AffiniPure 驴抗小鼠 IgG （H+L）（min X Bov， Ck， Gt， GP， Sy Hms， Hrs， 胡， Rb， 大鼠， Shp Sr Prot）	Jackson ImmunoResearch	715-175-151	驴抗小鼠（1：400 稀释），RRID：AB_2619678
Cy5 AffiniPure 驴抗绵羊 IgG （H+L）（min X Ck， GP， Sy Hms， Hrs， 胡， Ms， Rb， Rat Sr Prot）	Jackson ImmunoResearch	713-175-147	驴抗绵羊（1：400 稀释），RRID： AB_2340730
RNAscope 探针
Galanin 受体 1 寡核苷酸探针	ACDBio	448821-C1	靶向 bp 482 - 1669 （基因库参考：NM_008082.2）
甘氨酸转运蛋白 2 寡核苷酸探针	ACDBio	409741-C3	靶向 bp 925 - 2153 （基因库参考文献：NM_148931.3）
Phox2b 寡核苷酸探针	ACDBio	407861-C2	靶向 bp 1617 - 2790 （基因库参考文献：NM_008888.3）