rabbit anti nkcc2  (Cell Signaling Technology Inc)

Journal: The American Journal of Pathology

Article Title: Folliculin Deletion in the Mouse Kidney Results in Cystogenesis of the Loops of Henle via Aberrant TFEB Activation

doi: 10.1016/j.ajpath.2025.05.010

Figure Lengend Snippet: Flcn deletion causes cyst formation in the nephron, most prominent in the loop of Henle (LoH). A and B: Hematoxylin and eosin (H&E) staining of postnatal day 0 kidneys from control ( Flcn flox/flox ) and Flcn knockout (KO) ( Six2Cre ; Flcn flox/flox ) mice showing numerous cysts in both cortex and medulla. All KO mice ( N = 29) exhibited cystogenesis; representative images are presented. C and D: Immunofluorescence staining against LTL (proximal tubule), SLC12A1 (LoH), and WT1 (glomerular podocyte) confirming the presence of many cysts in the Flcn KO nephrons but not in controls. E and F: Higher magnification of C and D . White asterisks: Bowman's space; yellow asterisks: proximal tubule. G and H: Immunofluorescence staining against CDH1 SLC12A1 showing dilated LoH ( yellow arrows ). The lower left panels are higher magnification of the boxed regions , showing the irregularly curved lumen ( arrowheads ) with enlarged surrounding epithelia (SLC12A1 + /CDH1 + ) in Flcn KO but not in control LoH. The collecting ducts stained only with CDH1 are not dilated in either control or KO samples. Scale bars: 1 mm ( A and B ); 500 μm ( C and D ); 100 μm ( E–H ).

Article Snippet: The following primary antibodies were used: rabbit anti-SIX2 (#11562-1-AP; 1:400 dilution; Proteintech, Rosemont, IL), rat anti-KRT8 (1:10; Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti–E-cadherin (CDH1) (#610181; 1:100; BD, Franklin Lakes, NJ); rabbit anti-NKCC2 (SLC12A1) (#SPC-401D; 1:100; StressMarq Biosciences, Victoria, BC, Canada), goat anti-NKCC2 (SLC12A1) (#PAS-142445; 1:100; Invitrogen, Carlsbad, CA), rabbit anti-phospho-S6 ribosomal protein (Ser 240/244) (#5364; 1:100; Cell Signaling Technology, Danvers, MA), rabbit anti-WT1 (#GR 3270281-4; 1:100; Abcam, Cambridge, UK,), LTL (#B-132; 1:200; Vector Laboratories, Newark, CA), guinea pig anti-NEPHRIN (#GP-N2, 1:200; PROGEN, Heidelberg, Germany), rabbit anti-TFE3 (#HPA023881; 1:100; Sigma-Aldrich, St. Louis, MN), and rabbit anti-TFEB (#A303-673A, lot numbers 8 and 9; Bethyl Laboratories, Montgomery, TX).

Techniques: Staining, Control, Knock-Out, Immunofluorescence

Journal: The American Journal of Pathology

Article Title: Folliculin Deletion in the Mouse Kidney Results in Cystogenesis of the Loops of Henle via Aberrant TFEB Activation

doi: 10.1016/j.ajpath.2025.05.010

Figure Lengend Snippet: Uniform Manifold Approximation and Projection for Dimension Reduction plots of representative up-regulated genes in Flcn knockout (KO) loop of Henle (LoH). Uniform Manifold Approximation and Projection for Dimension Reduction plots and violin plots of representative up-regulated genes in the KO LoH. Atp6v0d2 , Rab27a , Lamtor1 , R r a gd , and Fnip2 are up-regulated in the homozygous KO LoH. Slc12a1 is used as an unaffected marker for LoH. Cluster numbers are presented in A. DT, distal tubule; NP, nephron progenitor; Pod, glomerular podocyte; PT, proximal tubule.

Article Snippet: The following primary antibodies were used: rabbit anti-SIX2 (#11562-1-AP; 1:400 dilution; Proteintech, Rosemont, IL), rat anti-KRT8 (1:10; Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti–E-cadherin (CDH1) (#610181; 1:100; BD, Franklin Lakes, NJ); rabbit anti-NKCC2 (SLC12A1) (#SPC-401D; 1:100; StressMarq Biosciences, Victoria, BC, Canada), goat anti-NKCC2 (SLC12A1) (#PAS-142445; 1:100; Invitrogen, Carlsbad, CA), rabbit anti-phospho-S6 ribosomal protein (Ser 240/244) (#5364; 1:100; Cell Signaling Technology, Danvers, MA), rabbit anti-WT1 (#GR 3270281-4; 1:100; Abcam, Cambridge, UK,), LTL (#B-132; 1:200; Vector Laboratories, Newark, CA), guinea pig anti-NEPHRIN (#GP-N2, 1:200; PROGEN, Heidelberg, Germany), rabbit anti-TFE3 (#HPA023881; 1:100; Sigma-Aldrich, St. Louis, MN), and rabbit anti-TFEB (#A303-673A, lot numbers 8 and 9; Bethyl Laboratories, Montgomery, TX).

Techniques: Knock-Out, Marker

Journal: The American Journal of Pathology

Article Title: Folliculin Deletion in the Mouse Kidney Results in Cystogenesis of the Loops of Henle via Aberrant TFEB Activation

doi: 10.1016/j.ajpath.2025.05.010

Figure Lengend Snippet: Transcription factor E3 (TFE3) and mammalian target of rapamycin complex 1 (mTORC1) are activated in Flcn knockout (KO) nephrons. A–D: In situ hybridization of Atp6v0d2 and Rab27a in Flcn flox/flox and KO kidneys. Slc12a1 is used as a marker for loop of Henle (LoH). The right panels are higher magnification of the boxed regions in the left panels . E–H: Immunofluorescence staining for TFE3, SLC12A1 (LoH), and LTL (proximal tubule) showing TFE3 nuclear reactivity in the cortex ( E and F ) but not in the medulla ( G and H ). Nuclear localization of TFE3 is detected in glomerular podocytes, Bowman's capsule ( white arrowheads ), and proximal tubules ( yellow arrowheads ) in Flcn KOs but not in LoH. The right panels are higher magnification of the boxed regions in the left panels . I–L: Immunofluorescence staining of phosphorylated S6 ribosomal protein (pS6), SLC12A1 (LoH), and LTL (proximal tubule) showing the increased pS6 in the LoH ( yellow arrows ) of Flcn KO kidneys. The right panels are higher magnification of the boxed regions in the left panels . Scale bars: 500 μm ( A–D ); 100 μm ( E–L ).

Article Snippet: The following primary antibodies were used: rabbit anti-SIX2 (#11562-1-AP; 1:400 dilution; Proteintech, Rosemont, IL), rat anti-KRT8 (1:10; Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti–E-cadherin (CDH1) (#610181; 1:100; BD, Franklin Lakes, NJ); rabbit anti-NKCC2 (SLC12A1) (#SPC-401D; 1:100; StressMarq Biosciences, Victoria, BC, Canada), goat anti-NKCC2 (SLC12A1) (#PAS-142445; 1:100; Invitrogen, Carlsbad, CA), rabbit anti-phospho-S6 ribosomal protein (Ser 240/244) (#5364; 1:100; Cell Signaling Technology, Danvers, MA), rabbit anti-WT1 (#GR 3270281-4; 1:100; Abcam, Cambridge, UK,), LTL (#B-132; 1:200; Vector Laboratories, Newark, CA), guinea pig anti-NEPHRIN (#GP-N2, 1:200; PROGEN, Heidelberg, Germany), rabbit anti-TFE3 (#HPA023881; 1:100; Sigma-Aldrich, St. Louis, MN), and rabbit anti-TFEB (#A303-673A, lot numbers 8 and 9; Bethyl Laboratories, Montgomery, TX).

Techniques: Knock-Out, In Situ Hybridization, Marker, Immunofluorescence, Staining

Journal: The American Journal of Pathology

Article Title: Folliculin Deletion in the Mouse Kidney Results in Cystogenesis of the Loops of Henle via Aberrant TFEB Activation

doi: 10.1016/j.ajpath.2025.05.010

Figure Lengend Snippet: Transcription factor EB (TFEB) is activated in Flcn knockout (KO) nephrons. A-D: Immunofluorescence staining for TFEB, SLC12A1 [loop of Henle (LoH)], and LTL (proximal tubule) showing TFEB nuclear reactivity in the cortex ( A and B ) and medulla ( C and D ) at postnatal day 0. Nuclear localization of TFEB is detected in proximal tubules ( yellow arrowheads ) and LoH ( yellow arrows ) in Flcn KOs. White arrowheads : Bowman’s capsule. The right panels are higher magnification of the boxed regions in the left panels . E and F: Immunofluorescence staining for TFEB, SLC12A1 (LoH), and KRT8 (collecting duct). The TFEB signal in the collecting duct remains unchanged. The right panels are higher magnification of the boxed regions in the left panels . Scale bars = 100 μm ( A–F ).

Article Snippet: The following primary antibodies were used: rabbit anti-SIX2 (#11562-1-AP; 1:400 dilution; Proteintech, Rosemont, IL), rat anti-KRT8 (1:10; Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti–E-cadherin (CDH1) (#610181; 1:100; BD, Franklin Lakes, NJ); rabbit anti-NKCC2 (SLC12A1) (#SPC-401D; 1:100; StressMarq Biosciences, Victoria, BC, Canada), goat anti-NKCC2 (SLC12A1) (#PAS-142445; 1:100; Invitrogen, Carlsbad, CA), rabbit anti-phospho-S6 ribosomal protein (Ser 240/244) (#5364; 1:100; Cell Signaling Technology, Danvers, MA), rabbit anti-WT1 (#GR 3270281-4; 1:100; Abcam, Cambridge, UK,), LTL (#B-132; 1:200; Vector Laboratories, Newark, CA), guinea pig anti-NEPHRIN (#GP-N2, 1:200; PROGEN, Heidelberg, Germany), rabbit anti-TFE3 (#HPA023881; 1:100; Sigma-Aldrich, St. Louis, MN), and rabbit anti-TFEB (#A303-673A, lot numbers 8 and 9; Bethyl Laboratories, Montgomery, TX).

Techniques: Knock-Out, Immunofluorescence, Staining

Journal: The American Journal of Pathology

Article Title: Folliculin Deletion in the Mouse Kidney Results in Cystogenesis of the Loops of Henle via Aberrant TFEB Activation

doi: 10.1016/j.ajpath.2025.05.010

Figure Lengend Snippet: Tfe3 deletion in the Flcn knockouts only ameliorates glomerular cysts. A: Scheme for the generation of Flcn ; Tfe3 double knockout (DKO) mice. Note that Tfe3 is located on the X chromosome. B and C: Immunofluorescence staining against TFE3, SLC12A1, and LTL showing the absence of TFE3 staining in the DKO mice. D and E: Hematoxylin and eosin (H&E) staining of postnatal day 0 kidneys from Flcn KO ( Six2Cre; Flcn flox/flox ) and Flcn ; Tfe3 DKO ( Six2Cre ; Flcn flox/flox ; Tfe3 del/Y ) mice showing amelioration of glomerular cysts in the DKO mice. Black arrows : Bowman’s space. Eleven DKO samples were examined; representative images are shown. F: Ratio of glomeruli with cysts to total glomeruli in H&E-stained sections. Sample numbers: 6 for Flcn KO; 8 for Flcn ; Tfe3 DKO. G–J: Immunofluorescence staining against LTL, SLC12A1, and WT1 highlighting proximal tubules, loop of Henle and glomerular podocytes. Glomerular cysts are reduced in DKOs ( yellow asterisks ), but cysts in the proximal tubules and loop of Henle are still present. The right panels are higher magnification of the boxed regions in the left panels . ∗ P < 0.05 ( P = 0.0340). Scale bars = 100 μm ( B – E and G– J ).

Article Snippet: The following primary antibodies were used: rabbit anti-SIX2 (#11562-1-AP; 1:400 dilution; Proteintech, Rosemont, IL), rat anti-KRT8 (1:10; Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti–E-cadherin (CDH1) (#610181; 1:100; BD, Franklin Lakes, NJ); rabbit anti-NKCC2 (SLC12A1) (#SPC-401D; 1:100; StressMarq Biosciences, Victoria, BC, Canada), goat anti-NKCC2 (SLC12A1) (#PAS-142445; 1:100; Invitrogen, Carlsbad, CA), rabbit anti-phospho-S6 ribosomal protein (Ser 240/244) (#5364; 1:100; Cell Signaling Technology, Danvers, MA), rabbit anti-WT1 (#GR 3270281-4; 1:100; Abcam, Cambridge, UK,), LTL (#B-132; 1:200; Vector Laboratories, Newark, CA), guinea pig anti-NEPHRIN (#GP-N2, 1:200; PROGEN, Heidelberg, Germany), rabbit anti-TFE3 (#HPA023881; 1:100; Sigma-Aldrich, St. Louis, MN), and rabbit anti-TFEB (#A303-673A, lot numbers 8 and 9; Bethyl Laboratories, Montgomery, TX).

Techniques: Double Knockout, Immunofluorescence, Staining

Journal: The American Journal of Pathology

Article Title: Folliculin Deletion in the Mouse Kidney Results in Cystogenesis of the Loops of Henle via Aberrant TFEB Activation

doi: 10.1016/j.ajpath.2025.05.010

Figure Lengend Snippet: Tfeb deletion in Flcn knockouts (KOs) restores all the phenotypes. A and B: Hematoxylin and eosin (H&E) staining of postnatal day 0 kidneys from Flcn KO and Flcn ; Tfeb double KO (DKO) mice showing reduction of cysts in both cortex and medulla ( black arrows ). Three DKO samples were examined; representative images are shown. C and D: Immunofluorescence staining showing the reduction of cysts in the DKO mice. Left panel: renal cortex; right panel: renal medulla. Yellow arrowheads : glomeruli; yellow asterisk: proximal tubule cyst; and yellow arrows: loop of Henle (LoH). Cysts are detected only in Flcn KO mice. E and F: Immunofluorescence staining against CDH1 and SLC12A1 showing amelioration of LoH abnormalities in the DKO. The right panels are higher magnification of the boxed regions in the left panels . G–J: Immunofluorescence staining of phosphorylated S6 ribosomal protein (pS6) showing its reduction in the DKO LoH. The right panels are higher magnification of the boxed regions in the left panels . Scale bars = 100 μm ( A–J ).

Article Snippet: The following primary antibodies were used: rabbit anti-SIX2 (#11562-1-AP; 1:400 dilution; Proteintech, Rosemont, IL), rat anti-KRT8 (1:10; Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti–E-cadherin (CDH1) (#610181; 1:100; BD, Franklin Lakes, NJ); rabbit anti-NKCC2 (SLC12A1) (#SPC-401D; 1:100; StressMarq Biosciences, Victoria, BC, Canada), goat anti-NKCC2 (SLC12A1) (#PAS-142445; 1:100; Invitrogen, Carlsbad, CA), rabbit anti-phospho-S6 ribosomal protein (Ser 240/244) (#5364; 1:100; Cell Signaling Technology, Danvers, MA), rabbit anti-WT1 (#GR 3270281-4; 1:100; Abcam, Cambridge, UK,), LTL (#B-132; 1:200; Vector Laboratories, Newark, CA), guinea pig anti-NEPHRIN (#GP-N2, 1:200; PROGEN, Heidelberg, Germany), rabbit anti-TFE3 (#HPA023881; 1:100; Sigma-Aldrich, St. Louis, MN), and rabbit anti-TFEB (#A303-673A, lot numbers 8 and 9; Bethyl Laboratories, Montgomery, TX).

Techniques: Staining, Immunofluorescence