European Journal of Histochemistry 2019; volume 63:2988

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Antigen retrieval pre-treatmentcauses a different expressionpattern of Cav3.2 in rat andmouse spinal dorsal hornXiao E Cheng,1,2 Long Xian Ma,2Xiao Jin Feng,1,2 Meng Ye Zhu,3Da Ying Zhang,3 Lin Lin Xu,1 Tao Liu1

1Center for Experimental Medicine, theFirst Affiliated Hospital of NanchangUniversity2Department of Anesthesiology, the FirstAffiliated Hospital of NanchangUniversity3Department of Pain Clinic, the FirstAffiliated Hospital of NanchangUniversity, China

AbstractCav3 channels consist of three isoforms,

Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3(α1I), which produce low-threshold spikesthat trigger burst firings in nociceptive neu-rons of the spinal dorsal horn (SDH) anddorsal root ganglion (DRG). AlthoughCav3.2 plays a crucial role in pathologicalpain, it is distribution in SDH still remainscontroversial. One study showed thatCav3.2 is ubiquitously expressed in neurons,but another study implied that Cav3.2 isexpressed restricted to astrocytes. To unrav-el these discrepancies, we used methods ofimmunohistochemistry either with or with-out antigen retrieval (AR) pre-treatment todetect Cav3 in SDH and DRG from both ratsand mice. Moreover, Cav3.2 mRNA wasdetected in mice SDH using in situhybridization. We found that the expressionpattern of Cav3.2 but not Cav3.1 and Cav3.3in SDH were largely different with or with-out AR pre-treatment, which showed a neu-ron-like and an astrocyte-like appearance,respectively. Double staining furtherdemonstrated that Cav3.2 was mainly co-stained with the neuronal marker NeuN inthe presence of AR but was with glial fibril-lary acidic protein (GFAP, marker for astro-cytes) in the absence of AR pre-treatment.Importantly, Cav3.2 mRNA was mainly co-localized with Cav3.2 but not GFAP.Together, our findings indicate that AR pre-treatment or not impacts the expression pat-tern of Cav3.2, which may make a signifi-cant contribution to the future study ofCav3.2 in SDH.

IntroductionT-type (Cav3) channels are low-voltage-

activated (LVA) calcium channels that areencoded by three α1 subunit genes: α1G(Cav3.1), α1H (Cav3.2), and α1I (Cav3.3),1

which are widely distributed in brain, spinalcord and dorsal root ganglion (DRG).2

Besides, immunohistochemical (IHC) stud-ies have demonstrated that the proteins ofCav3.1-3.3 are broadly expressed in theperipheral and central nervous system.3-7

The role of Cav3.2 in pathological pain hasbeen extensively studied by behavioral,electrophysiological, and molecular biolog-ical methods during the last decade. Forexample, the Western blot immunoassayfound that the expression of Cav3.2 wasincreased in DRG and spinal dorsal horn(SDH) from rodents of cystitis-related blad-der pain,8 paclitaxel-induced peripheralneuropathy,9 and loose ligatures of sciaticnerve induced neuropathic pain models,10

etc. Moreover, T-type currents recorded inDRG were enhanced in various animalmodels of pathological pain.11 Consistently,intrathecal injection of an antisenseoligonucleotide (targeted to the α1-subunitof Cav3.2, but not Cav3.1 or Cav3.3) or T-type channel blockers could alleviate patho-logical pain.12,13 The above results suggestthat Cav3.2 plays a crucial role in the mod-ulation of pathological pain. Therefore, it isimportant to identify the cell-type specificexpression of Cav3.2 channel in DRG andSDH. Up to now, the expression pattern ofCav3.2 in DRG is consensus, that is, Cav3.2is mainly distributed in small- and medium-diameter DRG neurons.5,9,13-18 However, inSDH, the expression pattern of Cav3.2 isdifferent. Chen et al. found that Cav3.2 wasrestricted to the neurons but not microgliaor astrocytes.5 In contrast, Li et al. foundthat Cav3.2 was co-localized to glial fibril-lary acidic protein (GFAP)-positive cells(marker of astrocyte) but not NeuN- (mark-er of neuron) or OX42-positive (marker ofmicroglia) cells.9 One possible explanationfor these differences might be thatformaldehyde fixation compromisesimmunoreactivity of Cav3.2 antigen by pre-venting contact between the epitopes andthe antibodies, which will affect the out-come of an IHC staining.19,20 To test thishypothesis, we investigated the IHC stain-ing of Cav3.2 in SDH and DRG sectionswith or without antigen retrieval (AR) pre-treatment which can unmask the antigens informaldehyde-fixed tissue sections. TheIHC staining of other two subtypes (Cav3.1and Cav3.3) were also tested as well. To fur-ther determine the nature of cell-type spe-cific expression of Cav3.2 protein, we alsoinvestigated the distribution of Cav3.2mRNA using in situ hybridization.

Together, our results demonstrated that ARpre-treatment is essential for the IHC exper-iment of Cav3.2 in SDH. These results mayprovide a theoretical basis for such kinds ofexperiments in the future.

Materials and Methods

AnimalsSprague-Dawley (SD) rats (5-8 w) and

wild type (WT) C57/BL6 mice (6-8 w) ofeither sex were obtained from the AnimalCenter of Nanchang University. Cav3.2knock-out (KO) mice (C57/BL6) were pur-chased from the Jackson Laboratory (BarHarbor, ME, USA). Animals were housedin controlled room temperature (RT, 22-25°C) and maintained on a 12-h light/darkcycle with water and food ad libitum. Allexperimental procedures were approved bythe Ethics Committee of NanchangUniversity.

Tissue collection and preparationThe rats and mice were deeply anes-

thetized by intraperitoneal injection of 1.5

Correspondence: Tao Liu, Center forExperimental Medicine, the First AffiliatedHospital of Nanchang University, 17Yongwaizheng Street, Nanchang 3330006,China. Tel. +86.791.88692139 - Fax: +86.791.88692139. E-mail: liutao1241@ncu.edu.cn

Key words: Cav3.2; immunohistochemistry;antigen retrieval; spinal dorsal horn.

Contributions: XEC, major experiments, man-uscript drafting; LXM, XJF, MYZ, DYZ,LLX, experiment assistance, data analysis;TL, study concept, data interpretation, manu-script revision.

Conflict of interest: The authors declare noconflict of interest.

Funding: This work was supported by theNational Natural Science Foundation of China(No. 31660289 to T.L. and 81560198 toD.Y.Z.) and the Outstanding Young PeopleFoundation of Jiangxi Province(20171BCB23091).

Received for publication: 18 October 2018.Accepted for publication: 20 January 2019.

This work is licensed under a CreativeCommons Attribution-NonCommercial 4.0International License (CC BY-NC 4.0).

©Copyright X.E. Cheng et al., 2019Licensee PAGEPress, ItalyEuropean Journal of Histochemistry 2019; 63:2988doi:10.4081/ejh.2019.2988

g/kg urethane and perfused transcardiallywith saline followed by cold 4%paraformaldehyde (PFA) in 0.1 M phos-phate buffer. The L4-L5 spinal cord seg-ments were removed and post-fixed in 4%PFA for 6 h, and then cryoprotected in a30% sucrose solution for 3 days. The L4and L5 DRGs were post-fixed in 4% PFAfor 6 h and cryoprotected in a 20% sucrosesolution for 12 h. After embedding in opti-mal cutting temperature medium, 30 μm(IHC staining) or 15 μm (in situ hybridiza-tion) transverse spinal cord sections and 15μm DRG sections were prepared using afreezing microtome (CM1950, Leica,Nussloch, Germany).

ImmunostainingAll the spinal cord and DRG sections

for IHC staining were first rinsed with 0.01M phosphate-buffered saline (PBS) threetimes. Then they were divided into no-AR(without AR pre-treatment) or AR (with ARpre-treatment) groups. Sections in ARgroups were subjected to heat-induced ARin a water bath (HH-2, China) at 98°C for10 min by using 0.01 M sodium citratebuffer (pH 6.0, Solarbio) and were cooled toRT. Next, the free-floating sections wereincubated in a blocking solution (0.3%Triton X-100, 1% albumin from bovineserum, and 1% normal donkey serum inPBS) for 30 min at RT to prevent nonspecif-ic staining and then were incubated in asolution containing primary antibodies forthree nights at 4˚C. Primary antibodies usedwere rabbit anti-Cav3.1-3.3 (1:200, Cat#ACC-021, Cat# ACC-025, Cat# ACC-009,Alomone), goat anti-Cav3.2 (1:50, Cat# sc-16261, Santa Cruz), guinea pig anti-NeuN(1:200, Cat# 266004, Synaptic Systems),goat anti-GFAP (1:2000, Cat# ab53554,Abcam). After washing, the sections wereincubated with appropriate secondary anti-bodies: donkey anti-rabbit Alexa Fluor 488(1:400, Cat# A-21206, Invitrogen), donkeyanti-goat Alexa Fluor 555 (1:400, Cat# A-21432, Invitrogen), donkey anti-guinea pigCy5 (1:400, Cat# 706-175-148, JacksonImmunoResearch) overnight at 4°C. Aftermounting the sections with Aqua-Poly/mount medium (Polysciences, Inc.,Warrington, PA), they were observed undera Zeiss LSM700 confocal microscope(Germany) with ZEN 2010 software. Thespecificity of Cav3.1-3.3 antibodies(Alomone) was tested by either omission orpre-absorption of primary antibodies withpeptide antigens using sections with ARpre-treatment. Moreover, the specificity ofanti-Cav3.2 (Alomone) was also confirmedby the Cav3.2 KO mice. Co-localizationimages of Cav3.2 with other antibodies weretaken with a 63x oil objective at a 0.5xzoom. The parameters for acquiring the

images, such as the number of recordingpixels, electrical shutter speed, gain, andpinhole were kept unchanged throughoutthe whole experiment process. The quantifi-cation of co-localization was assessed bythe ZEN 2010 software as our previousstudy.21

In situ hybridizationThe method for in situ hybridization

was modified from a previous publication.15

The Cav3.2 probe (5’-ACAAUGCCAU-CAAAGAUGUUGUAGGGGUUCC-GAAUG-3’) was designed for mouseCacna1h gene (NM_021415.4) and waslabeled at 5’-end with digoxigenin. Briefly,spinal cord sections were treated with pro-teinase K (20 μg/mL) for 20 min and fixedin 4% PFA for 15 min at RT. After that, thesections were rinsed with PBS containing0.1% Tween-20 (PBST). Next, they weretreated with 0.25% acetic anhydride in 0.1M triethanolamine (pH 8.0) and rinsed withPBST three times. Following this, sectionswere incubated in pre-hybridization solu-tion (50% formamide, 5×SSC, 0.1%Tween-20, 0.3 mg/mL yeast tRNA, and5×Denhardt’s Solution) for 4 h at 37°C, andthen incubated in the hybridization solution(pre-hybridization solution plus digoxin-labeled probe) for 16 h at 37°C. Afterhybridization, the sections were washedthree times with 2X saline-sodium citrate,containing 0.1% Tween-20 (SSCT) at 37°C(15 min each) and one time with 1X SSCTand 0.5x SSCT at RT, respectively.Afterward, the sections were blocked with0.5% sheep serum (Solarbio) for 1.5 h andincubated with alkaline phosphatase-conju-gated anti-digoxigenin antibody (RocheDiagnostics) overnight at 4°C. Then theywere incubated with a mixture of nitrobluetetrazolium chloride (NBT, SangonBiotech) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP, Sangon Biotech) for 1~3h for color development. In addition, tocompare the results of the in situ hybridiza-tion with that of the IHC, partial sectionswere further incubated with anti-Cav3.2(Alomone) and GFAP as well as 4’,6-Diamidino-2-Phenylindole (DAPI, 1:1000,Cat# D9542, Sigma, sections were incubat-ed with DAPI for 10 min at RT beforemounting) and were visualized by anFSX100 microscope equipped with a digitalcamera system (Olympus, Japan) or theZeiss LSM700 confocal microscope.

Statistical analysis SPSS version 17.0 (SPSS Inc, Chicago,

IL, USA) was used for statistical analysis.All data are expressed as mean ± SEM.Shapiro–Wilk test was used to assess thenormality of data. Differences betweengroups were compared using unpaired

Student’s t-test. P<0.05 was considered sta-tistically significant.

Results

Immunolabeling of Cav3 isoforms inSDH and DRG of SD rats with orwithout AR pre-treatment

Although the distributions of Cav3.1,Cav3.2, and Cav3.3 mRNAs have been iden-tified in both SDH and DRG,2 IHC studiesin these areas were mostly focused onCav3.2. However, hyperalgesia could alsobe observed in Cav3.1 null mice,22 suggest-ing Cav3.1 might contribute to the sensoryperception of pain as well. Therefore, wefirst investigated the expression of Cav3.1,Cav3.2, and Cav3.3 isoforms in SDH andDRG with or without AR pre-treatment. Asshown in Figure 1, regardless of whethersections were processed with (right) orwithout (left) AR, Cav3.1-immunoreactivity(IR) showed a punctate staining pattern inSDH (Figure 1 A,D) and Cav3.3-IR was pri-marily distributed around the neuron-likemembranes (Figure 1 C,F). However, wefound a significantly different expressionpattern of Cav3.2-IR in sections processedwith AR from those without AR pre-treat-ment. For sections lacking AR pre-treat-ment, Cav3.2-IR showed a glial cell-likeappearance (Figure 1B), which is similar tothe results of Li et al.9 However, for thosesections with AR pre-treatment, Cav3.2-IRshowed a neuron-like appearance (Figure1E), which was in line with the findings ofChen et al.5 Next, we investigated whetherAR-caused different IHC staining exists inother tissues, such as DRG, which is mostlystudied for the mechanisms of pain. Asshown in Figure 1 G-L, Cav3.1, Cav3.2, andCav3.3-IR were highly expressed at neuron-like membranes and cytoplasm. In addition,unlike the observations of Cav3.2-IR inSDH, AR pre-treatment did not affect theexpression pattern of Cav3.2 in DRG.Together, these findings demonstrate thatAR pre-treatment altered the IHC stainingresults of Cav3.2 in rat SDH but not DRG,without any effect on Cav3.1 and Cav3.3 iso-forms.

Immunolabeling of Cav3 isoforms inSDH and DRG of mice with or with-out AR pre-treatment

Since the Cav3.2-IR showed a differentIHC staining pattern in rat SDH, we wonderwhether this difference is due to the speciesof animal. Besides rat, mouse is the mostcommonly used animal species. Therefore,we next performed the same experiment onmice SDH. We found that Cav3.1- (Figure 2A,D) and Cav3.3-IR (Figure 2 C,F) were

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[European Journal of Histochemistry 2019; 63:2988] [page 25]

similar in mice SDH sections with or withoutAR pre-treatment. However, the results ofCav3.2-IR (Figure 2 B,E) were affected byAR pre-treatment, which is highly similar tothat of the rats. AR pre-treatment led to theCav3.2-IR exhibited a neuron-like appear-ance in mice SDH, too. However, no differ-ences of Cav3.1- and Cav3.3-IR in SDH fromboth rat and mouse were observed whetherpre-treated with AR or not.

Discrepancies of Cav3.2-IR from twodifferent sources of antibodies withor without AR pre-treatment

Anti-Cav3.1-Cav3.3 antibodies used inthe above study were all from the AlomoneCompany. We were curious whether the dif-ferent results of Cav3.2-IR in SDH wouldhave been the same if an alternative com-mercial anti-Cav3.2 antibody was used.

Therefore, we next compared the Cav3.2-IRin SDH by using two different commercialsources of anti-Cav3.2 (Alomone vs SantaCruz). As shown in Figure 3 A-C, withoutAR pre-treatment, the IR of the two sourcesof Cav3.2 antibodies were similar in ratSDH sections, which both showed a glialcell-like appearance and a substantial co-localization. However, for those sectionswith AR pre-treatment, the IR of anti-Cav3.2 antibody from Alomone was mostlyexpressed in neuron-like cells (Figure 3D),while there was no change for the IR ofanti-Cav3.2 antibody from Santa Cruzwhich still shows a glial cell-like appear-ance (Figure 3E). Furthermore, after pre-treating with AR, the extent of co-stainingwas significantly decreased (Figure 3F).These data suggested that AR pre-treatment

has a different effect on Cav3.2-IR from var-ious commercial sources of antibodies.

Co-localization of Cav3.2 with GFAPand NeuN in sections of SDH pre-treated with AR or not

Above results suggested that immuno-staining of Cav3.2 (Alomone) has neuron-likeand glial-cell like appearances with or with-out AR pre-treatment, respectively. To furtherconfirm the cell-type specific expression ofCav3.2, we next co-stained Cav3.2 withGFAP and NeuN. As shown in Figure 4A-E,it is clear that without AR pre-treatment,Cav3.2 was largely co-stained with GFAP butnot NeuN. In contrast, with AR pre-treat-ment, Cav3.2 was major co-stained withNeuN but not GFAP (Figure 4F-J).Quantitative analysis (Figure 4K) demon-strated that the co-localized ratio of Cav3.2

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Figure 1. Expression of Cav3.1-3.3 isoforms (Alomone) in SDH and DRG of SD rats with or without AR pre-treatment. Representativeconfocal images of Cav3.1 (A), Cav3.2 (B), and Cav3.3 (C) in SDH without AR pre-treatment (No AR). Representative confocal imagesof Cav3.1 (D), Cav3.2 (F), and Cav3.3 (F) in SDH with AR pre-treatment (AR). Representative images of Cav3.1 (G), Cav3.2 (H), andCav3.3 (I) in DRG without AR pre-treatment (No AR). Representative images of Cav3.1 (J), Cav3.2 (K), and Cav3.3 (L) in DRG withAR pre-treatment (AR).

Original Paper

Figure 2. Expression of Cav3.1-3.3 isoforms (Alomone) in mice SDH sections pre-treated with AR or not. Representative images ofCav3.1 (A), Cav3.2 (B), and Cav3.3 (C) in SDH without AR pre-treatment (no AR). Representative images of Cav3.1 (D), Cav3.2 (E), andCav3.3 (F) in SDH with AR pre-treatment (AR).

Figure 3. Expression of anti- Cav3.2 from two companies in rat SDH with or without AR pre-treatment. A) Representative image of Cav3.2(Alomone, green) in SDH without AR pre-treatment (No AR). B) Representative image of Cav3.2 (Santa Cruz, red) in SDH without AR pre-treatment (No AR). C) Merged images of (A) and (B). D) Representative image of Cav3.2 (Alomone, green) in SDH with AR pre-treatment (AR).E) Representative image of Cav3.2 (Santa Cruz, red) in SDH with AR pre-treatment (AR). F) Merged images of (D) and (E).

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with GFAP in sections without AR pre-treat-ment was higher (30.4±4.4% vs 9.4±0.4%,n=7, P<0.01) than that of the sections pre-treated with AR. The results for the co-local-ization of Cav3.2 with NeuN were vice versa(12.4±0.7% vs 28.2±1.3%, n=7, P<0.001). Incontrol experiments by omitting the primaryantibodies or pre-absorbing the primary anti-bodies with peptide antigens, no IHC signalwas detected (Supplementary Figures 1 and2). Moreover, we tested the specificity ofCav3.2 antibody (Alomone) by using theSDH sections with AR pre-treatment fromWT and Cav3.2 KO mice. Strong Cav3.2-IR

was observed in SDH of WT mice (Figure4L) but not Cav3.2 KO mice (Figure 4M),which demonstrated the specificity of Cav3.2antibody (Alomone). Furthermore, westernblotting using SDH tissues from miceimplied that Cav3.2 (Alomone) is expressedin WT mouse rather than KO mouse, sug-gesting its fine specificity for western blot(Supplementary Figure 3). Together, theseresults suggested that AR pre-treatmentcaused the co-localization of Cav3.2 shiftedfrom GFAP to NeuN.

To identify the nature distribution patternof Cav3.2, we next performed the in situ

hybridization experiment to study the expres-sion of Cav3.2 mRNA, which showed that theCav3.2 mRNA positive stain cells have a neu-ron-like appearance (Figure 5A). The sec-tions of in situ hybridization were furtherprocessed to IHC co-stain for Cav3.2, GFAP,and DAPI. Figure 5 B-G showed that most ofthe Cav3.2 mRNA positive cells are also anti-Cav3.2-positive but not GFAP-positive cells.For conclusion, these results demonstratedthat the expression pattern of Cav3.2-IR withAR pre-treatment is closer to that of Cav3.2mRNA, suggesting that the Cav3.2 positivecells should be neurons but not astrocytes.

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Figure 4. Expression pattern of Cav3.2 (Alomone) in rat SDH with or without AR pre-treatment. Representative images of Cav3.2 (A,green), GFAP (B, red), NeuN (C, magenta), merged images of Cav3.2 and GFAP (D), and merged images of Cav3.2 and NeuN (E) inSDH without AR pre-treatment (No AR). Representative images of Cav3.2 (F, green), GFAP (G, red), NeuN (H, magenta), mergedimages of Cav3.2 and GFAP (I), and merged images of Cav3.2 and NeuN (J) in SDH with AR pre-treatment (AR). K) Quantitative co-localization results of Cav3.2-IR with NeuN- and GFAP-IR. Representative images of Cav3.2 (Alomone) in SDH from wild-type mice(L) and Cav3.2 knock-out (KO) mice (M). **P<0.01, ***P<0.001.

DiscussionThe immunohistochemistry technique

is used to detect cell or tissue antigens.23

Principal factors affecting the outcome ofIHC studies include the following: i) tissuefixation and processing, ii) unmasking of

epitopes, and iii) sensitivity of the detectionsystem.24 Formaldehyde is one of the mostpopular fixatives due to its low cost, ease ofpreparation, and because of its well mor-phologic detail with few artifacts. However,formaldehyde fixation could mask or dam-age some binding sites of antibodies, which

might lead to the loss of IR.25,26 AR pre-treatment, which is mainly based on high-temperature heating of tissues, is an effec-tive method to overcome these disadvan-tages.27,28 Since 2007, genetic linkage stud-ies have implicated Cav3.2 gene coded pro-tein as a pain modulation related protein.

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Figure 5. Expression pattern of Cav3.2 mRNA in mouse SDH. A) Microscopic images of a DIG-labeled Cav3.2 probe for in situhybridization in mice SDH. Triple-labeling of in situ hybridization sections for Cav3.2 mRNA (B, purple) and the following IHC stain-ing of Cav3.2 (C, green, Alomone), GFAP (D, red), DAPI (E, magenta), merged images of Cav3.2 and GFAP (F), and merged images ofCav3.2 and DAPI (G). Note that Cav3.2 mRNA is expressed in Cav3.2-positive cells (arrows) but not GFAP-positive astrocytes.

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However, there were disagreements as tothe expression pattern of Cav3.2 in SDH ofSD rat.5,9 We therefore raised the hypothesisthat AR pre-treatment might account forthis difference. To test this hypothesis, weobserved the effect of AR pre-treatment onIHC staining of Cav3.1-3.3 in rat SDH andDRG as well as mouse SDH.

Cav3 channels can be activated aroundresting membrane potential and are inacti-vated within a few tens of milliseconds.29

Hence, they are also known as transientlyopen calcium channels. In the nervous sys-tem, Cav3 channels are involved in the reg-ulation of neuronal excitability, thus theyare linked to the pathogenesis of variousneurological disorders, including chronicpain. In multiple animal models of neuro-pathic pain, Cav3 was upregulated in DRGand SDH.30-32 Among the three Cav3 iso-forms, Cav3.2 is the most concerned sub-type with neuropathic pain.12 Consistentwith previous studies showing that Cav3.2was mainly distributed in small- to medi-um-sized DRG neurons,14,15 our data foundthat Cav3.2, as well as Cav3.1 and Cav3.3,had similar expression pattern in DRG withor without AR pre-treatment. Interestingly,previous studies showed that the distribu-tion pattern of Cav3.2 in SDH is controver-sial. Chen et al. found that, in L5/6 spinalnerve ligation pain model, the expression ofCav3.2 (Santa Cruz, rabbit) in SDH of SDrats was upregulated seven days after nerveligation.5 Moreover, they found that Cav3.2is expressed in neurons (co-localize withNeuN) but not astrocytes (co-localize withGFAP). However, in SDH of SD rats suf-fered from paclitaxel-induced peripheralneuropathy, the Cav3.2 (Alomone, rabbit)expression was increased and co-localizedto GFAP positive but not NeuN positivecells.9 These distinct results may be due tothe antibodies they used were from differentcompanies. Thus, we investigated theCav3.2 expression in SDH of SD rats withthe antibodies from the above two compa-nies. Differently from Chen et al.’s study(Santa Cruz - rabbit),5 our results found thatCav3.2-IR from Santa Cruz (goat) showed aglia-like appearance and was irrelevant toAR pre-treatment. This difference might bedue to the different sourced anti-Cav3.2.However, Santa Cruz does not provide rab-bit-sourced Cav3.2 which is out of the mar-ket. Currently, only a mouse-sourced Cav3.2is available. Therefore, we performed theIHC staining of Cav3.2 using a rabbit-sourced anti-Cav3.2 (C1868) from Sigma-Aldrich (data not shown). Whereas, theCav3.2-IR (Sigma-Aldrich) displayed apunctate staining with or without AR pre-treatment, suggesting the source of Cav3.2has little effect on Cav3.2-IR. In contrast,AR pre-treatment influenced the results of

Cav3.2-IR from Alomone (rabbit). Ourtriple-labeling experiments found thatCav3.2 (Alomone) was co-localized withNeuN but not GFAP after AR pre-treatment,further demonstrating that AR pre-treatmentimpacts the results of Cav3.2-IR in SDH.

Given that mice are also the commonlyused rodents for pain animal models, wenext observed the effect of AR on Cav3.2(Alomone) on SDH sections from mouseand got the same results. A recent studydemonstrated that Cav3.2 was co-stainedwith PKCγ-containing interneurons in theCav3.2-GFP knock-in mice, suggestingCav3.2 is expressed in neurons.6 In line withthis study, our IHC results of Cav3.2-IR inmice showed that under the pre-treatment ofAR, Cav3.2-IR displayed the neuron-likeappearance.

Although one previous report describedCav3.2 (α1H) mRNA was mostly expressedin the outermost layers (layers 1-2) ofSDH,2 our present findings indicate thatCav3.2 mRNA is present throughout thegray matter of spinal cord. Since the sec-tions for in situ hybridization included sodi-um citrate and heat pre-treatment, post-IHCof Cav3.2 protein (Alomone) from in situhybridization sections largely matched thedistribution of Cav3.2 mRNA. Furthermore,our and previous electrophysiological stud-ies have revealed the presence of T-typechannels in SDH neurons,33-35 which highlysuggested that Cav3.2 is expressed in neu-rons. These data suggested that AR pre-treatment might be necessary for specificantigens, including Cav3.2.

Taken together, we have demonstratedthat AR pre-treatment affect the Cav3.2-IRin rat and mouse SDH and we believe thatour findings make a significant contributionto the future research of Cav3.2 distributionin SDH from both rats and mice.

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