Journal: The Journal of Biological Chemistry

Article Title: The RNA methyltransferase NSUN2 catalyzes 5-methylcytosine (m 5 C) on IL1B mRNA to promote transcript stability

doi: 10.1016/j.jbc.2026.111290

Figure Lengend Snippet: YBX1, acting as an m 5 C reader, collaborates with NSUN2 to facilitate the expression of inflammatory cytokines in DPCs. A , total RNA was purified from DPCs stimulated with LPS. RT-PCR analyzed the mRNA expression of the m 5 C reader ( YBX1 , YTHDF2 , and ALYREF) and inflammatory cytokines ( IL-1β and IL-6 ). β-actin served as a loading control. Data are means ± SD, n = 3. B , representative IHC staining of YBX1 expression in normal (n = 6) and pulpitis (n = 5) teeth . The scale bars represent 50 μm. P: pulp tissue; D: dentine. C , the expression of YBX1 in dental pulp detected by IHC was quantified as the average optical density (AOD) using ImageJ software and IHC Toolbox plugin. D , measurement of YBX1 mRNA expression analyzed by RT-qPCR in normal (n = 7) and pulpitis (n = 8) teeth . β-actin served as a loading control. E–G , DPCs were transfected with either Myc-tagged YBX1 or empty vector (control) plasmids. The transcriptional levels of IL1B and IL6 ( E ), as well as CXCL10 ( F ) and CCL2 ( G ), were detected by RT-PCR. β-actin served as a loading control. Data are means ± SD, n = 3 or 4. H , western blot confirmed the overexpression efficiency of YBX1. The anti-Myc antibody was used to detect the Myc-tagged fusion protein. GAPDH served as a loading control. I–K , DPCs were treated with LPS or PBS after transfection with either siYBX1 or siNC. I , knockdown efficiency of siYBX1 was confirmed by western blot. GAPDH served as a loading control. J and K , RT-PCR was employed to illustrate the effect of YBX1 knockdown on the mRNA levels of IL1B and CXCL10 ( J ) as well as IL6 and CCL2 ( K ) in DPCs. β-actin served as a loading control. Data are means ± SD, n = 3 or 4. L–N The interaction between YBX1 and IL1B transcripts were analyzed by RNA immunoprecipitation (RIP) using anti-YBX1 antibody, with normal rabbit IgG as the isotype control. L and N , immunoprecipitated RNA was analyzed by RT-PCR. L , endogenous IL1B transcripts bound by YBX1. N , exogenous GFP-IL1B transcripts bound by YBX1. The histogram below showed the percentage of GFP-ILB mRNA co-immunoprecipitated with YBX1 relative to Input levels. Data are means ± SD, n = 3. M and N , immunoprecipitated YBX1 protein was confirmed by western blot. O , the mRNA half-life of IL1B in DPCs transfected with siYBX1. siNC was used as a negative control and β-actin served as a loading control. Data are means ± SD, n = 4. P–R , DPCs with or without NSUN2 overexpression were transfected with siYBX1 or siNC. The expression levels of IL1B and CCL2 ( P ), as well as IL6 and CXCL10 ( Q ), were detected by RT-PCR. β-actin served as a loading control. Data are means ± SD, n = 3 or 4. R , the overexpression of NSUN2 and the knockdown efficiency of siYBX1 were analyzed by western blot. GAPDH served as a loading control. S , RIP-PCR analysis of the interaction between YBX1 and IL1B mRNA in NSUN2 knockdown and control DPCs. T , schema of the mechanism by which NSUN2 collaborating with YBX1 promotes the progression of dental pulp inflammation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ALYREF, Aly/REF export factor; CCL2, C-C motif chemokine ligand 2; CXCL10, C-X-C motif chemokine ligand 10; DPCs, dental pulp cells; IL1B, interleukin 1 beta; LPS, lipopolysaccharides; m5C, 5-methylcytosine; NSUN, NOP2/Sun RNA methyltransferase; RT-PCR, semiquantitative reverse transcription PCR; RT-qPCR, real-time quantitative reverse transcription PCR; YBX1, Y-box binding protein 1.

Article Snippet: After blocked in 5% defatted milk, the membranes were incubated with the following antibodies: rabbit monoclonal anti-NSUN2 (#ab259941, 1:2000, Abcam), rabbit monoclonal anti-YBX1 (#ab76149, 1:2000, Abcam), mouse monoclonal anti-YBX1 (#TA806283, 1:1000, Origene Technologies), rabbit anti-FLAG antibody (#20543, 1:2000, Proteintech), rabbit anti-MYC antibody (#10828, 1:2000, Proteintech), and mouse anti-GAPDH (#sc-47724, 1:1000, Santa Cruz Biotechnology).

Techniques: Expressing, Purification, Reverse Transcription Polymerase Chain Reaction, Control, Immunohistochemistry, Software, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Over Expression, Knockdown, RNA Immunoprecipitation, Immunoprecipitation, Negative Control, Reverse Transcription, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The RNA methyltransferase NSUN2 catalyzes 5-methylcytosine (m 5 C) on IL1B mRNA to promote transcript stability

doi: 10.1016/j.jbc.2026.111290

Figure Lengend Snippet: YBX1, acting as an m 5 C reader, collaborates with NSUN2 to facilitate the expression of inflammatory cytokines in DPCs. A , total RNA was purified from DPCs stimulated with LPS. RT-PCR analyzed the mRNA expression of the m 5 C reader ( YBX1 , YTHDF2 , and ALYREF) and inflammatory cytokines ( IL-1β and IL-6 ). β-actin served as a loading control. Data are means ± SD, n = 3. B , representative IHC staining of YBX1 expression in normal (n = 6) and pulpitis (n = 5) teeth . The scale bars represent 50 μm. P: pulp tissue; D: dentine. C , the expression of YBX1 in dental pulp detected by IHC was quantified as the average optical density (AOD) using ImageJ software and IHC Toolbox plugin. D , measurement of YBX1 mRNA expression analyzed by RT-qPCR in normal (n = 7) and pulpitis (n = 8) teeth . β-actin served as a loading control. E–G , DPCs were transfected with either Myc-tagged YBX1 or empty vector (control) plasmids. The transcriptional levels of IL1B and IL6 ( E ), as well as CXCL10 ( F ) and CCL2 ( G ), were detected by RT-PCR. β-actin served as a loading control. Data are means ± SD, n = 3 or 4. H , western blot confirmed the overexpression efficiency of YBX1. The anti-Myc antibody was used to detect the Myc-tagged fusion protein. GAPDH served as a loading control. I–K , DPCs were treated with LPS or PBS after transfection with either siYBX1 or siNC. I , knockdown efficiency of siYBX1 was confirmed by western blot. GAPDH served as a loading control. J and K , RT-PCR was employed to illustrate the effect of YBX1 knockdown on the mRNA levels of IL1B and CXCL10 ( J ) as well as IL6 and CCL2 ( K ) in DPCs. β-actin served as a loading control. Data are means ± SD, n = 3 or 4. L–N The interaction between YBX1 and IL1B transcripts were analyzed by RNA immunoprecipitation (RIP) using anti-YBX1 antibody, with normal rabbit IgG as the isotype control. L and N , immunoprecipitated RNA was analyzed by RT-PCR. L , endogenous IL1B transcripts bound by YBX1. N , exogenous GFP-IL1B transcripts bound by YBX1. The histogram below showed the percentage of GFP-ILB mRNA co-immunoprecipitated with YBX1 relative to Input levels. Data are means ± SD, n = 3. M and N , immunoprecipitated YBX1 protein was confirmed by western blot. O , the mRNA half-life of IL1B in DPCs transfected with siYBX1. siNC was used as a negative control and β-actin served as a loading control. Data are means ± SD, n = 4. P–R , DPCs with or without NSUN2 overexpression were transfected with siYBX1 or siNC. The expression levels of IL1B and CCL2 ( P ), as well as IL6 and CXCL10 ( Q ), were detected by RT-PCR. β-actin served as a loading control. Data are means ± SD, n = 3 or 4. R , the overexpression of NSUN2 and the knockdown efficiency of siYBX1 were analyzed by western blot. GAPDH served as a loading control. S , RIP-PCR analysis of the interaction between YBX1 and IL1B mRNA in NSUN2 knockdown and control DPCs. T , schema of the mechanism by which NSUN2 collaborating with YBX1 promotes the progression of dental pulp inflammation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ALYREF, Aly/REF export factor; CCL2, C-C motif chemokine ligand 2; CXCL10, C-X-C motif chemokine ligand 10; DPCs, dental pulp cells; IL1B, interleukin 1 beta; LPS, lipopolysaccharides; m5C, 5-methylcytosine; NSUN, NOP2/Sun RNA methyltransferase; RT-PCR, semiquantitative reverse transcription PCR; RT-qPCR, real-time quantitative reverse transcription PCR; YBX1, Y-box binding protein 1.

Article Snippet: Small interfering RNAs (siRNAs) targeting human NSUN2 and YBX1 were synthesized by Sangon Biotech.

Techniques: Expressing, Purification, Reverse Transcription Polymerase Chain Reaction, Control, Immunohistochemistry, Software, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Over Expression, Knockdown, RNA Immunoprecipitation, Immunoprecipitation, Negative Control, Reverse Transcription, Binding Assay

Journal: iScience

Article Title: YBX1 promotes angiogenesis after myocardial infarction by stabilizing HIF1α mRNA via m 6 A signaling

doi: 10.1016/j.isci.2026.114810

Figure Lengend Snippet: YBX1 is upregulated in endothelial cells after MI and in hypoxic endothelial cells (A) UMAP of single-nucleus RNA sequencing (snRNA-seq) data ( n = 191,795 nuclei) annotated into major cardiac cell types. (B–D) Sample origin overlay for the same atlas: (B) Normal (green) versus Disease/MI (red) combined, (C) Normal subset only, and (D) Disease/MI subset only. (E) Violin plots of YBX1 expression across major cardiac cell types, showing increased expression in endothelial cells and reduced expression in cardiomyocytes in Disease/MI compared with Normal. (F–H) Feature plots of YBX1 expression in (F) all nuclei, (G) Normal, and (H) Disease/MI samples, demonstrating predominant localization of YBX1 signal within endothelial cell clusters. (I) Left: Representative immunofluorescence images of mouse hearts 28 days after MI or Sham surgery showing YBX1 (green) and CD31 (red) with DAPI (blue). The peri-infarct border region is indicated. Scale bars, 20 μm. Right: Quantification of YBX1 + cells among CD31 + endothelial cells in Sham and MI hearts ( n = 5). (J) Western blot analysis of YBX1 expression in HUVECs under normoxic or hypoxic conditions, with densitometric quantification normalized to β-actin ( n = 3). Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01 (unpaired t test).

Article Snippet: After natural cooling to room temperature, sections were blocked with ready-to-use normal goat serum (Elabscience, E-IR-R110) at 37°C for 30 minutes, followed by overnight incubation at 4°C with anti-YBX1 antibody (proteintech, 20339-1-AP, 1:300).

Techniques: RNA Sequencing, Expressing, Immunofluorescence, Western Blot

Journal: iScience

Article Title: YBX1 promotes angiogenesis after myocardial infarction by stabilizing HIF1α mRNA via m 6 A signaling

doi: 10.1016/j.isci.2026.114810

Figure Lengend Snippet: YBX1 promotes endothelial viability, proliferation, migration, and tube formation in vitro (normoxia) (A) CCK-8 time-course showing that YBX1 overexpression (YBX1) increases, whereas YBX1 knockdown (si-YBX1) decreases, HUVEC viability compared with respective negative controls (NC), ( n = 5). (B) Wound-healing assays at 0, 24, and 48 h with quantification of migration area/cells. Scale bars, 100 μm, ( n = 5). (C) EdU incorporation images and quantification showing enhanced proliferation with YBX1 and reduced proliferation with si-YBX1. DAPI marks nuclei. Scale bars, 50 μm, ( n = 5). (D) Matrigel tube-formation images and quantification (relative tube length). Scale bars, 200 μm, ( n = 5). (E) Transwell migration images and quantification. Scale bars, 50 μm, ( n = 6). Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (unpaired t test).

Article Snippet: After natural cooling to room temperature, sections were blocked with ready-to-use normal goat serum (Elabscience, E-IR-R110) at 37°C for 30 minutes, followed by overnight incubation at 4°C with anti-YBX1 antibody (proteintech, 20339-1-AP, 1:300).

Techniques: Migration, In Vitro, CCK-8 Assay, Over Expression, Knockdown

Journal: iScience

Article Title: YBX1 promotes angiogenesis after myocardial infarction by stabilizing HIF1α mRNA via m 6 A signaling

doi: 10.1016/j.isci.2026.114810

Figure Lengend Snippet: YBX1 promotes angiogenesis in vascular endothelial cells under hypoxia (A) CCK-8 time-course showing that YBX1 overexpression increases, whereas YBX1 knockdown decreases, HUVEC viability under hypoxic conditions ( n = 5). (B) Wound-healing assays at 0, 24, and 48 h under hypoxia with quantification of migration area. Scale bars, 100 μm ( n = 5). (C) EdU incorporation images and quantification showing enhanced proliferation with YBX1 and reduced proliferation with si-YBX1 under hypoxia. DAPI marks nuclei. Scale bars, 50 μm ( n = 5). (D) Matrigel tube-formation images and quantification (relative tube length) under hypoxia. Scale bars, 200 μm ( n = 5). (E) Transwell migration images and quantification under hypoxia. Scale bars, 50 μm ( n = 6). Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (unpaired t test).

Article Snippet: After natural cooling to room temperature, sections were blocked with ready-to-use normal goat serum (Elabscience, E-IR-R110) at 37°C for 30 minutes, followed by overnight incubation at 4°C with anti-YBX1 antibody (proteintech, 20339-1-AP, 1:300).

Techniques: CCK-8 Assay, Over Expression, Knockdown, Migration

Journal: iScience

Article Title: YBX1 promotes angiogenesis after myocardial infarction by stabilizing HIF1α mRNA via m 6 A signaling

doi: 10.1016/j.isci.2026.114810

Figure Lengend Snippet: Overexpression of YBX1 improves cardiac injury after MI (A) Survival rates of Sham and MI mice injected with AAV9 over a 4-week follow-up period. (B) Heart-to-weight ratio in mice ( n = 6). (C and D) Representative M-mode echocardiograms and corresponding left ventricular internal dimension at end-diastole (LVIDd) and end-systole (LVIDs) in mice subjected to Sham or MI treatment ( n = 10). (E) Representative cross-sectional Masson’s trichrome–stained images and quantification of scar size 28 days after MI. Scale bar, 500 μm ( n = 5). (F) Representative H&E-stained cardiac sections from Sham and MI hearts with or without YBX1 overexpression 28 days post-MI. Scale bars, 500 μm (upper panels) and 50 μm (lower panels). (G and H) Immunohistochemistry for CD31 + vessels and α-SMA + smooth muscle/pericytes, and immunostaining for cTnT + cardiomyocytes at 28 days post-MI. Scale bar, 50 μm. ( n = 5). Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (One-way ANOVA).

Article Snippet: After natural cooling to room temperature, sections were blocked with ready-to-use normal goat serum (Elabscience, E-IR-R110) at 37°C for 30 minutes, followed by overnight incubation at 4°C with anti-YBX1 antibody (proteintech, 20339-1-AP, 1:300).

Techniques: Over Expression, Injection, Staining, Immunohistochemistry, Immunostaining

Journal: iScience

Article Title: YBX1 promotes angiogenesis after myocardial infarction by stabilizing HIF1α mRNA via m 6 A signaling

doi: 10.1016/j.isci.2026.114810

Figure Lengend Snippet: Knockdown YBX1 aggravates cardiac injury after MI (A) Survival rates of sham and MI mice injected with AAV9 over a 4-week follow-up period. (B) Heart-to-weight ratio in mice ( n = 6). (C, D) Representative M-mode echocardiograms and corresponding left ventricular internal dimension at end-diastole (LVIDd) and end-systole (LVIDs) in mice subjected to Sham or MI treatment ( n = 10). (E) Representative Masson’s trichrome–stained cross-sectional images and quantification of scar size in hearts 28 days after MI .Scale bar, 500 μm. ( n = 5). (F) Representative hematoxylin and eosin (H&E)–stained heart sections showing myocardial histopathological changes 28 days after MI. Scale bars, 500 μm (upper panels) and 50 μm (lower panels). (G and H) Immunohistochemistry for CD31 + vessels and α-SMA + smooth muscle/pericytes, and immunostaining for cTnT + cardiomyocytes at 28 days post-MI. Scale bar, 50 μm ( n = 5). Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01 (One-way ANOVA).

Article Snippet: After natural cooling to room temperature, sections were blocked with ready-to-use normal goat serum (Elabscience, E-IR-R110) at 37°C for 30 minutes, followed by overnight incubation at 4°C with anti-YBX1 antibody (proteintech, 20339-1-AP, 1:300).

Techniques: Knockdown, Injection, Staining, Immunohistochemistry, Immunostaining

Journal: iScience

Article Title: YBX1 promotes angiogenesis after myocardial infarction by stabilizing HIF1α mRNA via m 6 A signaling

doi: 10.1016/j.isci.2026.114810

Figure Lengend Snippet: HIF1α mediates the angiogenesis of YBX1 (A) RNA-seq volcano plot after YBX1 knockdown showing differentially expressed genes (DEGs). (B) KEGG enrichment of DEGs highlights pathways linked to angiogenesis and stress responses, including the HIF1α signaling pathway. (C) GSEA shows significant enrichment of the HIF1α signaling pathway in control versus YBX1-deficient cells, indicating pathway attenuation upon YBX1 loss. (D and E) EdU (red) with DAPI (blue) and merged images under normoxia (D) and hypoxia (E) for four groups. Scale bars, 50 μm. ( n = 4). (F and G) Wound-healing assays at 0, 24, and 48 h under normoxia (F) and hypoxia (G) with quantification of migration area (%), Scale bars, 100 μm. ( n = 5). (H and I) Tube-formation (top) and Transwell migration (bottom) under normoxia (H) and hypoxia (I) with quantification of relative tube length and migrated cells (%). Scale bars, 200 μm (tube formation) and 50 μm (Transwell). ( n = 4–5). Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01 (One-way ANOVA).

Article Snippet: After natural cooling to room temperature, sections were blocked with ready-to-use normal goat serum (Elabscience, E-IR-R110) at 37°C for 30 minutes, followed by overnight incubation at 4°C with anti-YBX1 antibody (proteintech, 20339-1-AP, 1:300).

Techniques: RNA Sequencing, Knockdown, Control, Migration

Journal: iScience

Article Title: YBX1 promotes angiogenesis after myocardial infarction by stabilizing HIF1α mRNA via m 6 A signaling

doi: 10.1016/j.isci.2026.114810

Figure Lengend Snippet: YBX1 affects the mRNA stability of HIF1α (A) RNA dot blot analysis of m 6 A levels methylation modification in cardiac tissue. Methylene blue (MB) staining served as a loading control. (B) The statistical graph shows the quantitative analysis of RNA dot blot result ( n = 3). (C–E) Potential m 6 A modification sites of HIF1α were predicted by SRAMP ( n = 3). (F) MeRIP-qPCR analysis of m 6 A enrichment of HIF1α in HUVECs ( n = 3). (G) mRNA half-life of HIF1α in NC and siYBX1 -HUVECs ( n = 6). (H) Co-IP and western blot detection of the interaction between YBX1 and IGF2BP1/IGF2BP3. (I and J) YBX1 RIPqPCR analysis showing YBX1 binding to HIF1α mRNA in NC or siIGF2BP1/siIGF2BP3 HUVECs ( n = 3). Data are presented as mean ± SEM.∗ p < 0.05, ∗∗ p < 0.01 (unpaired t test).

Article Snippet: After natural cooling to room temperature, sections were blocked with ready-to-use normal goat serum (Elabscience, E-IR-R110) at 37°C for 30 minutes, followed by overnight incubation at 4°C with anti-YBX1 antibody (proteintech, 20339-1-AP, 1:300).

Techniques: Dot Blot, Methylation, Modification, Staining, Control, Co-Immunoprecipitation Assay, Western Blot, Binding Assay

Journal: iScience

Article Title: YBX1 promotes angiogenesis after myocardial infarction by stabilizing HIF1α mRNA via m 6 A signaling

doi: 10.1016/j.isci.2026.114810

Figure Lengend Snippet: Overexpression of YBX1 improves cardiac injury after MI (A) Survival rates of Sham and MI mice injected with AAV9 over a 4-week follow-up period. (B) Heart-to-weight ratio in mice ( n = 6). (C and D) Representative M-mode echocardiograms and corresponding left ventricular internal dimension at end-diastole (LVIDd) and end-systole (LVIDs) in mice subjected to Sham or MI treatment ( n = 10). (E) Representative cross-sectional Masson’s trichrome–stained images and quantification of scar size 28 days after MI. Scale bar, 500 μm ( n = 5). (F) Representative H&E-stained cardiac sections from Sham and MI hearts with or without YBX1 overexpression 28 days post-MI. Scale bars, 500 μm (upper panels) and 50 μm (lower panels). (G and H) Immunohistochemistry for CD31 + vessels and α-SMA + smooth muscle/pericytes, and immunostaining for cTnT + cardiomyocytes at 28 days post-MI. Scale bar, 50 μm. ( n = 5). Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (One-way ANOVA).

Article Snippet: The sections were stained overnight with an anti-YBX1 antibody (proteintech, 20339-1-AP, 1:300), anti-YBX1 antibody (proteintech, 20339-1-AP, 1:300), anti-αSMA antibody (proteintech, 14395-1-AP, 1:300), anti-cTnT antibody (proteintech, 68300-1-Ig 1:300), and anti-CD31 antibody (Servicebio, GB11063-2-100, 1:300), followed by incubation with a secondary antibody conjugated to fluorochrome for 1 hour.

Techniques: Over Expression, Injection, Staining, Immunohistochemistry, Immunostaining

Journal: iScience

Article Title: YBX1 promotes angiogenesis after myocardial infarction by stabilizing HIF1α mRNA via m 6 A signaling

doi: 10.1016/j.isci.2026.114810

Figure Lengend Snippet: Knockdown YBX1 aggravates cardiac injury after MI (A) Survival rates of sham and MI mice injected with AAV9 over a 4-week follow-up period. (B) Heart-to-weight ratio in mice ( n = 6). (C, D) Representative M-mode echocardiograms and corresponding left ventricular internal dimension at end-diastole (LVIDd) and end-systole (LVIDs) in mice subjected to Sham or MI treatment ( n = 10). (E) Representative Masson’s trichrome–stained cross-sectional images and quantification of scar size in hearts 28 days after MI .Scale bar, 500 μm. ( n = 5). (F) Representative hematoxylin and eosin (H&E)–stained heart sections showing myocardial histopathological changes 28 days after MI. Scale bars, 500 μm (upper panels) and 50 μm (lower panels). (G and H) Immunohistochemistry for CD31 + vessels and α-SMA + smooth muscle/pericytes, and immunostaining for cTnT + cardiomyocytes at 28 days post-MI. Scale bar, 50 μm ( n = 5). Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01 (One-way ANOVA).

Article Snippet: The sections were stained overnight with an anti-YBX1 antibody (proteintech, 20339-1-AP, 1:300), anti-YBX1 antibody (proteintech, 20339-1-AP, 1:300), anti-αSMA antibody (proteintech, 14395-1-AP, 1:300), anti-cTnT antibody (proteintech, 68300-1-Ig 1:300), and anti-CD31 antibody (Servicebio, GB11063-2-100, 1:300), followed by incubation with a secondary antibody conjugated to fluorochrome for 1 hour.

Techniques: Knockdown, Injection, Staining, Immunohistochemistry, Immunostaining